Detection of degradative enzymes and biomolecules in bodily fluids

ABSTRACT

Provided herein are compositions useful in detecting degradative enzymes and biomolecules in bodily fluid samples.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/567,758, filed Dec. 11, 2014, which in turn is a divisional of U.S.patent application Ser. No. 13/936,882, filed Jul. 8, 2013, now issuedas U.S. Pat. No. 8,940,866, which in turn is a divisional of U.S. patentapplication Ser. No. 12/866,732, filed Dec. 1, 2010, now issued as U.S.Pat. No. 8,507,218, which is a U.S. National Stage of InternationalApplication No. PCT/US2009/033584, filed Feb. 9, 2009, which claims thebenefit of U.S. Provisional Application No. 61/027,308, filed on Feb. 8,2008, each of which is herein incorporated by reference in its entiretyfor all purposes.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file 48537-502D03US_ST25.TXT, created onNov. 11, 2015, 11,240 bytes, machine format IBM-PC, MS-Windows operatingsystem, is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Increasing evidence suggests that there is an association between aninflammatory cascade and physiological shock [1, 2], diabetes [3, 4],cardiovascular diseases [5-10], acute cerebral stroke [11-15],Alzheimer's chronic disease [16], and various other diseases. Thiscascade is accompanied by activation of cells, expression ofpro-inflammatory genes, down regulation of anti-inflammatory genes,attachment of leukocytes to the endothelium, elevated permeability ofthe endothelium, thrombosis, mast cell degranulation, apoptosis, growthfactor release, and other events [17]. This evidence has opened up greatopportunities in medicine to develop a variety of new anti-inflammatoryinterventions in an increasing number of diseases. Recent researchdesigned to determine the origin of the trigger mechanisms of theinflammatory cascade in shock, multi-organ failure, and other diseasesshow that there exists an enhanced level of degradative enzymes that aretargeted towards a variety of autologous proteins, protein structures,lipids, and lipid structures [1, 18-20]. This enzyme activity is notblocked to the same level as in non-disease control samples.

In shock, for example, digestive enzymes (e.g. chymotrypsin, trypsin,elastase, lipase, nuclease) synthesized in the pancreas find entry intothe wall of the intestine [1]. Physiological shock is a life-threateningcardiovascular complication with high mortality that occurs insituations associated with trauma, including burns, surgery, ischemia,and sepsis. These traumas cause a reduced blood flow in the intestine,which in turn triggers an increased epithelial and endothelialpermeability. This allows pancreatic enzymes to enter systemiccirculation via the portal vein and/or intestinal lymphatics, where theyproduce a chain of events, including the production of inflammatorymediators (toxic protein fragments), inflammation, self-digestion oftissues, multi-organ failure, and eventually death. These enzymes havethe ability to degrade almost all biological tissues and molecules, and,when exposed to the body during shock, lead to auto-digestion of matrixproteins and tissue cells in the intestinal wall and to the productionof inflammatory mediators, which in turn further enhances the level ofinflammation. Detection of these proteases in the blood can thereforediagnose a patient for the early stages of physiological shock, as wellas for chronic and acute inflammation. It is also believed that thedetection of lipases, amylases, and nucleases may be important fordiagnosing these diseases. Furthermore the detection of proteases,lipases, amylases, and nucleases may also be important for many otherdiseases, including heart disease and cancer i.e., pancreatic cancer inparticular.

As an additional example, diabetes is a disease characterized byexcessive blood glucose levels [21]. Too much glucose in the blood cancause acute complications such as hypoglycemia, ketoacidosis andnonketotic hyperosmolar coma, as well as chronic complications such ascardiovascular disease, chronic renal failure, retinal damage(potentially resulting in blindness), several types of nerve damage, andmicrovascular damage (which can lead to impotence and poor healing).Glucose uptake from the blood is stimulated by the hormone insulin.Diabetes occurs when this hormone can't be synthesized by the body (typeI) or when the body has resistance or decreased sensitivity to it (typeII). For the latter case, recent evidence has shown that one particularpathogenesis of this insulin resistance may be proteolytic cleavage ofthe extracellular α-subunit of the insulin receptor by matrixmetalloproteases (MMPs) [22]. It was shown, that spontaneouslyhypertensive rats (SHR) had significantly elevated MMP-9 protein levelsin SHR microvessels, and elevated levels of leukocytes compared tonormotensive Wistar-Kyoto rats. Furthermore, in-vivo micro-zymographyshowed enhanced cleavage by MMP-1,9 that co-localized with MMP-9 and wasblocked by metal chelation. Using an antibody against the extracellulardomain of the insulin receptor, this study further showed reduceddensity of the insulin receptor-α and a corresponding elevation ofglucose and glycated hemoglobin in the blood, compared to thenormotensive control. Treating the SHR with a broad spectrum MMPinhibitor, doxycycline, reversed all of these aforementioned trends. Inother studies by Lee [23] and by Derosa [24], it was shown that therewere elevated MMP-2, 9 levels in diabetic patients versus healthypatients. Together, all of these results show that one or more MMPs maybe responsible for cleavage of the insulin receptor-alpha andcorresponding insulin resistance, which in turn leads to type-2diabetes. Detecting these matrix metalloproteases can therefore diagnosea developing insulin resistance during the early stages of type 2diabetes.

Previous protease detection substrates and devices have includedFluorogenic Substrates [27, 28], Chromogenic Substrates [25, 26],FRET-Based Substrates [29, 30], EnzChek Assays, ImmunohistochemicalAssays, Fluorescence Polarization [31, 32] and Zymography [33]. Theseare protease assays based on cleavage of specific short amino-acidsequences designed to detect specific protease activity. The existingprotease detection kits are based on 96 well plates with relativelylarge sample size (0.1 to 0.3 ml) each and they are not generallydesigned to detect cleavage of specific proteases. In general, thesesubstrates are FRET type peptides which when cleaved by the proteaseproduce an enhanced fluorescent signal or change in the fluorescentemission wavelength. Fluorescent PepTag® substrates are separated by gelelectrophoresis after hydrolysis [34, 35]. These assays do not allow thedetection of proteases directly in blood, cannot be separated easilyfrom blood and plasma components, and are not useful for clinicaldiagnostics.

Other devices, systems and substrates for the detection of proteinkinases and proteases have been designed [36, 37], however they appearnot to be useful for rapid clinical diagnostic applications for thereasons discussed above.

Since the discovery of the link between the inflammatory cascade andphysiological shock, diabetes, and potentially numerous other diseases,there have not been any detection platforms developed that could performmultiplex measurements of the clinical levels of disease related enzymesdirectly in blood or plasma. Therefore, there is a clear need for therapid and quantitative detection of key disease related enzymes informats that utilize minimal sample size and sample preparation. Thepresent invention addresses these and other needs in the art.

BRIEF SUMMARY OF THE INVENTION

Provided herein are methods, kits and compositions useful in detectingdegradative enzymes and biomolecules in bodily fluid samples.

In one aspect, a method of detecting a degradative enzyme in a bodilyfluid sample is provided. The method includes the step of contacting thebodily fluid sample with a negatively charged degradative enzymesubstrate or neutral degradative enzyme substrate. The degradativeenzyme is allowed to react with the negatively charged degradativeenzyme substrate or neutral degradative enzyme substrate thereby forminga positively charged degradative enzyme product. The positively chargeddegradative enzyme product is electrophoretically separated from thenegatively charged degradative enzyme substrate or neutral degradativeenzyme substrate. The separated positively charged degradative enzymeproduct is detected thereby detecting the degradative enzyme in thebodily fluid sample.

In another aspect, a peptide sequence is provided including at least oneof the sequences of SEQ ID NOs:1-18 or conservative amino acidsubstitutions thereof.

In another aspect, a kit for detecting a degradative enzyme in a crudebodily fluid sample is provided. The kit includes a negatively chargeddegradative enzyme substrate or neutral degradative enzyme substrate.

In another aspect, a method is provided for detecting a biomolecule in abodily fluid sample. The method includes contacting a bodily fluidsample with a first detection antibody and a second positively chargedantibody to form a detectable positively charged biomolecule conjugate.The detectable positively charged biomolecule conjugate iselectrophoretically separated from negatively charged endogenousmaterial present in the bodily fluid sample. The detectable positivelycharged biomolecule conjugate is detected thereby detecting thebiomolecule in the fluid sample.

In another aspect, a method of detecting a nucleic acid in a bodilyfluid sample is provided. The method includes the steps of contactingthe bodily fluid sample with a first detection nucleic acid and a secondpositively charged nucleic acid to form a detectable positively chargednucleic acid conjugate. The detectable positively charged nucleic acidconjugate is electrophoretically separated from negatively chargedendogenous material present in the bodily fluid sample. The detectablepositively charged nucleic acid conjugate is detected thereby detectingthe nucleic acid in the bodily fluid sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B. Populations of healthy patients and those with a specificdisease have Gaussian distributions of activity of a specific protease.FIG. 1A: A threshold is set in a diagnostic test to designate whichrange of activities will produce a positive result. FIG. 1B: If thethreshold is set lower to increase sensitivity of detection, this willincrease false positives and hence lower specificity.

FIG. 2. A healthy and diseased population are not well resolved by theactivity of a single protease (2D plot) but are better resolved by theactivities of multiple proteases (3D plot).

FIGS. 3A-3C. Design and synthesis of the net charge differentiatingpeptide substrates (NCDPS), cleavage, separation and detection. (FIG.3A) Chymotrypsin substrate labeled with a fluorophore and a net chargeof −1. The fluorescently labeled cleavage product has a net charge of+2. (FIG. 3B) Without an electric field, uncleaved substrate(background) and cleaved product (signal) are unresolved. (FIG. 3C)After application of an electric field, the uncleaved substrate andfluorescently labeled cleavage product migrate in opposite directions.The fluorescently labeled cleavage product can be detected by afluorescent detector. Sequences:Ac-N-Asp-Gly-Asp-Ala-Gly-Tyr-Ala-Gly-Leu-Arg-Gly-Ala-Gly (SEQ ID NO:19);Ac-N-Asp-Gly-Asp-Ala-Gly-Tyr (SEQ ID NO:20); Ala-Gly-Leu-Arg-Gly-Ala-Gly(SEQ ID NO:21).

FIGS. 4A-4C. Cleavage and separation of the NCDPS substrate and productfragments in whole blood.

FIG. 5. Peptide substrates for detecting inflammatory cascade enzymes.Each sequence carries cleavage sites at defined locations to promotespecific detection. Different fluorescent substrates allow simultaneousdetection of multiple enzymes. Sequence:Gly-Glu-Gly-Ala-Phe-Gly-Ala-Arg-Gly (SEQ ID NO:22).

FIG. 6. Multiplexing NCDPS.

FIG. 7. Design and synthesis of the net charge differentiating peptidesubstrate (NCDPS) with a streptavidin Quantum Dot or fluorescentnanoparticle label. Sequence:Gly-Glu-Gly-Ala-Phe-Gly-Ala-Arg-Gly-(biotin) (SEQ ID NO:23);Gly-Glu-Gly-Ala-Phe-Gly-Ala-Arg-Gly (SEQ ID NO:22).

FIG. 8. 3D or tertiary NCDPS substrates. Two substrates are shown forchymotrypsin and trypsin that have the same amino acid sequence, butdifferent tertiary structures.

FIG. 9. Design for net charge changing amylase substrate.

FIG. 10. Design for net charge changing lipase substrate.

FIG. 11. Design for net charge changing nuclease substrate.

FIG. 12. Scheme for net charge changing double antibody assay.

FIG. 13. Schematic of an enzyme separation and detection system in amicrotiter plate format.

FIGS. 14A-14B. Schematic of an enzyme separation and detection system ina focusing/smart gel format.

FIG. 15. Schematic of an enzyme separation and detection system in amicroelectrode array format.

FIG. 16. Schematic of an enzyme separation and detection system in amicrofluidic format.

FIG. 17. Schematic of an enzyme separation and detection system whichaccelerates the reaction of the protease with the NCDPS substrates.

FIG. 18. Schematic of an enzyme separation and detection system whichaccelerates the reaction of the protease with immobilized NCDPSsubstrates.

FIG. 19. Reduction of electrophoretic mobility of Streptavidin QuantumDot-Biotinyl-Gln-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 derivative (SEQID NO:24) (Example 1).

FIG. 20. Electrophoretic separation of α-chymotrypsin and trypsin in1×PBS buffer using a NCDPS (Example 2).

FIG. 21. Enzyme activity standard curves for detection of α-chymotrypsinand trypsin in 1×PBS buffer using a NCDPS (Example 2).

FIG. 22. Electrophoretic separation of α-chymotrypsin and trypsin inhuman plasma using a NCDPS (Example 3).

FIG. 23. Enzyme activity standard curves for detection of α-chymotrypsinand trypsin in human plasma using a NCDPS (Example 3).

FIG. 24. Agarose gel electrophoresis patterns of α-chymotrypsin (gel I)and trypsin (gel II) cleavage products in whole rat blood (Example 4).

FIG. 25. Enzyme activity standard curves generated from the detection ofα-chymotrypsin (curve I) and trypsin (curve II) cleavage products inwhole rat blood (Example 4).

FIGS. 26A-26B. Electrophoretic pattern of theAcetyl-N-Asp-Gly-Asp-Ala-Gly-Tyr-/-Ala-Gly-Leu-/-Arg-/-Gly-Ala-Gly-BodipyFL (SEQ ID NO:25) reacted with bovine pancreatic alpha-chymotrypsin in1×PBS (left half) and whole rat blood (right half). Legend: FIG. 26A(upper); FIG. 26B (lower).

FIG. 27. Non-specific binding activity of the NCDPS substrate.

FIG. 28. Stacking gel focusing of the NCDPS substrate.

FIG. 29. High density capillary gel format for focusing the NCDPSsubstrate.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Provided herein are, inter alia, methods for the design, synthesis anduse of degradative enzyme substrates, such as net charge differentiatingpeptide substrates (NCDPS), for the rapid detection of disease relatedenzymes in bodily fluids such as blood, plasma and other biologicalsamples. NCDPS's can be labeled with a variety of detection moietieswhich include but are not limited to organic fluorophores, quantum dots,fluorescent nanoparticles, fluorescent proteins, dendrimericnanoparticles, and chemiluminescent and electrochemical labels. TheseNCDPS's can be used for the highly specific and sensitive detection ofthe key disease related enzymes, including but not limited to proteases,matrix metalloproteases, lipases, amylases, nucleases and proteinkinases associated with shock, inflammatory responses and many otherdiseases. The substrates may be specifically designed for use in blood,plasma and other crude (e.g. un-processed) biological samples.

In some embodiments, the methods allow the rapid identification ofspecific disease related enzymes with little or no sample preparation.For example, upon cleavage by a specific enzyme, degradative enzymesubstrates produce a cleavage product which has an overall net chargethat is different from the original peptide substrate (e.g. neutral ornegatively charged peptide substrates). The cleaved substrates (e.g.positively charged peptide substrates) may be specifically designed tobe rapidly separated from the substrate itself, as well as components ofa bodily fluid such as blood cells and other background blood and plasmacomponents (hemoglobin, albumin, etc.). In certain embodiments, thesubstrate design allows the cleaved fluorescent peptide productfragments to be selectively concentrated by unique electrophoreticfocusing/smart gels and microgel devices, as well as in microtiter plateand lab-on-a-chip devices providing greatly enhanced signal detection.Also provided are novel research and clinical diagnostic systems,devices and signal detection components which are synergistic with theNCDPS's, allowing the substrates to be used for the rapid detection ofproteases and other enzymes directly in blood, plasma and other relevantsamples.

In certain embodiments of the methods provided herein, electric fieldsare used to rapidly separate and concentrate fluorescent labeled peptidefragments after cleavage by a specific degradative enzyme. The processmay be designed to greatly increase the sensitivity and specificity fordetecting specific proteases and other enzyme (e.g. lipases, amylases,nucleases, kinases, etc.) directly in blood or plasma. Where low levelsof the target degradative enzyme or biomolecules are present, highlysensitive and specific methods are required. In some embodiments, highlysensitive and specific methods are required for patient monitoring andpoint of care (POC) applications. Cleavage of degradative enzymesubstrates results in a change in the net charge on the complex. Thecleaved products can be separated and concentrated from the intactpeptide substrate by application of a directed electrophoretic field.Subsequent detection may be performed with a high sensitivityfluorescent, luminescent or electronic detection component device. Themethods may employ a micro-titer plate, microarray, and microfocusinggel formats as research and diagnostic platform systems for a variety ofapplications.

II. Definitions

A “subject” refers to an animal. In some embodiments, the subject is amammalian subject such as a human, non-human primate (e.g. rat, mouse orother rodent), or domesticated animal (e.g. dog, horse, cat, pig).

Generally, a “sample” represents a mixture containing or suspected ofcontaining an analyte to be measured in an assay. Samples which can betypically used in the methods of the invention include bodily fluidssuch as blood, which can be anti-coagulated blood as is commonly foundin collected blood specimens, plasma, urine, semen, saliva, cellcultures, tissue extracts and the like.

The term “specific binding” refers to binding between two molecules suchas a ligand and a receptor and is characterized by the ability of amolecule (ligand) to associate with another specific molecule (receptor)in the presence of many other diverse molecules. Specific binding of aligand to a receptor is also evidenced by reduced binding of adetectably labeled ligand to the receptor in the presence of excess ofunlabeled ligand (i.e. a binding competition assay).

The term “antibody” (Ab) refers to a polypeptide with a framework regionfrom an immunoglobulin gene or fragments thereof that specifically bindsand recognizes an antigen. The recognized immunoglobulin genes includethe kappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genesImmunoglobulin light chains are classified as either kappa or lambda,whereas immunoglobulin heavy chains are classified as gamma, mu, alpha,delta, or epsilon. The immunoglobulin heavy chains define theimmunoglobulin classes (isotypes), IgG, IgM, IgA, IgD and IgE,respectively. Typically, the antigen-binding region of an antibody willbe most critical in specificity and affinity of binding. Antibodies canbe polyclonal or monoclonal, derived from serum, a hybridoma orrecombinantly cloned, and can also be chimeric, primatized, orhumanized.

An example of an immunoglobulin (antibody) structural unit includes atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). Disulfide bonds connect the heavy chainand the light chain of each individual pair. Further, the two heavychains of each binding pair are connected through a disulfide bond inthe hinge region. Each heavy and light chain has two regions, a constantregion and a variable region. The constant region of the heavy chain isidentical in all antibodies of the same isotype, but differs inantibodies of different isotypes. The variable region located at theN-terminus of the heavy and the light chain includes about 100 to 110 ormore amino acids and is primarily responsible for antigen recognition.The terms variable light chain (V_(L)) and variable heavy chain (V_(H))refer to these light and heavy chains, respectively.

Antibodies exist, for example as intact immunoglobulins or as a numberof well-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab, which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into a Fab monomer. The Fab monomer is essentially Fab with partof the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993).While various antibody fragments are defined in terms of the digestionof an intact antibody, one of skill will appreciate that such fragmentsmay be synthesized de novo either chemically or by using recombinant DNAmethodology. Thus, the term antibody, as used herein, also includesantibody fragments either produced by the modification of wholeantibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990)).

“Polypeptide” refers to a polymer in which the monomers are amino acidsand are joined together through amide bonds, alternatively referred toas a peptide. When the amino acids are α-amino acids, either thel-optical isomer or the d-optical isomer can be used. Additionally,unnatural amino acids, for example, β-alanine, phenylglycine andhomoarginine are also included. Commonly encountered amino acids thatare not gene-encoded may also be used in the present invention. All ofthe amino acids used in the present invention may be either the d- orl-isomer. The l-isomers are generally preferred. In addition, otherpeptidomimetics are also useful in the present invention. For a generalreview, see, Spatola, A. F., in Chemistry and Biochemistry of AminoAcids, Peptides and Proteins, B. Weinstein, eds., Marcel Dekker, NewYork, p. 267 (1983).

The phrase “amino acid” as used herein refers to any of the twentynaturally occurring amino acids as well as any modified amino acids.Modifications can include natural processes such as posttranslationalprocessing, or chemical modifications which are known in the art.Modifications include, but are not limited to, phosphorylation,ubiquitination, acetylation, amidation, glycosylation, covalentattachment of flavin, ADP-ribosylation, cross linking, iodination,methylation, and the like.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form, andcomplements thereof. The term encompasses nucleic acids containing knownnucleotide analogs or modified backbone residues or linkages, which aresynthetic, naturally occurring, and non-naturally occurring, which havesimilar binding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides.

A “nucleoside analog” as used herein is defined in more detail below andincludes analogs of ribonucleosides and deoxyribonucleosides and thetriphosphates thereof. As described above, they are naturally occurringor non-naturally occurring, and derived from natural sources orsynthesized. These monomeric units are nucleoside analogs (or“nucleotide” analogs if the monomer is considered with reference tophosphorylation). For instance, structural groups are optionally addedto the sugar or base of a nucleoside for incorporation into anoligonucleotide, such as a methyl or allyl group at the 2′-O position onthe sugar, or a fluoro group which substitutes for the 2′-O group, or abromo group on the nucleoside base. The phosphodiester linkage, or“sugar-phosphate backbone” of the oligonucleotide analog is substitutedor modified, for instance with methyl phosphonates, phosphorothioates,dithiophosphates, boranophosphates, or O-methyl phosphates.

The terms “NCDPS”, “NCDAS”, “NCDLS”, “NCDNS” and “NCDPKS” stand for netcharge differentiating protease substrate, net charge differentiatingamylase substrate, net charge differentiating lipase substrate, netcharge differentiating nuclease substrate and net charge differentiatingprotein kinase substrate, respectively. NCDPS, NCDAS, NCDLS, NCDNS andNCDPKS are characterized by a specific net charge. For instance, forNCDPS the net charge can be either neutral, negative (e.g. −1) orpositive (e.g. +1). The net charge confers a particular migratorypotential to the NCDPS upon exposure to an electric field. Therefore,NCDPS with a negative net charge will migrate towards the anode, whereasNCDPS with a positive net charge will migrate towards the cathode of anelectrochemical device. However, neutral NCDPS exhibit no substantialmigration potential in an electrical field. Examples for other chargedifferentiating enzyme substrates as disclosed herein are kinasesubstrates, methytransferase substrates and aminotransferase substrates.In general any substrate specific for an enzyme present in a bodilyfluid sample can be used in the present methods and devices.

The term “non-amino acid groups” includes any biological molecule otherthan an amino acid. Examples for non-amino acid groups are monomers,polymers, oligomers, small molecules, hyaluronic acid, RNA/DNAfragments, glucosaminoglycan, polyethylene glycols, polyacrylamides,succinic acid, aminobutyric acid.

The term “isomeric amino acid” includes amino acids that are present inan optical isomeric form other than the L form. Examples are D-aminoacids which are naturally found in proteins by exotic sea dwellingorganisms, and are abundant components of the peptidoglycan cell wallsof bacteria.

The term “reactive functional group” as used herein includes, forexample:

(a) carboxyl groups and various derivatives thereof including, but notlimited to, N-hydroxysuccinimide esters, N-hydroxybenztriazole esters,acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl,alkenyl, alkynyl and aromatic esters;

(b) hydroxyl groups which can be converted to esters, ethers, aldehydes,etc.

(c) haloalkyl groups wherein the halide can be later displaced with anucleophilic group such as, for example, an amine, a carboxylate anion,thiol anion, carbanion, or an alkoxide ion, thereby resulting in thecovalent attachment of a new group at the site of the halogen atom;

(d) dienophile groups which are capable of participating in Diels-Alderreactions such as, for example, maleimido groups;

(e) aldehyde or ketone groups such that subsequent derivatization ispossible via formation of carbonyl derivatives such as, for example,imines, hydrazones, semicarbazones or oximes, or via such mechanisms asGrignard addition or alkyllithium addition;

(f) sulfonyl halide groups for subsequent reaction with amines, forexample, to form sulfonamides;

(g) thiol groups, which can be converted to disulfides or reacted withacyl halides;

(h) amine or sulfhydryl groups, which can be, for example, acylated,alkylated or oxidized;

(i) alkenes, which can undergo, for example, cycloadditions, acylation,Michael addition, etc;

(j) epoxides, which can react with, for example, amines and hydroxylcompounds; and

(k) phosphoramidites and other standard functional groups useful innucleic acid synthesis.

The reactive functional groups can be chosen such that they do notparticipate in, or interfere with, the crosslinking reactions disclosedherein. Alternatively, a reactive functional group can be protected fromparticipating in the crosslinking reaction by the presence of aprotecting group. Those of skill in the art will understand how toprotect a particular functional group from interfering with a chosen setof reaction conditions. For examples of useful protecting groups, SeeGreene et al., Protective Groups in Organic Synthesis, John Wiley &Sons, New York, 1991.

III. Methods of Detecting Degradative Enzymes

In one aspect, a method of detecting a degradative enzyme in a bodilyfluid sample is provided. The method includes the step of contacting thebodily fluid sample with a negatively charged degradative enzymesubstrate or neutral degradative enzyme substrate. The degradativeenzyme is allowed to react with the negatively charged degradativeenzyme substrate or neutral degradative enzyme substrate thereby forminga positively charged degradative enzyme product. The positively chargeddegradative enzyme product is electrophoretically separated from thenegatively charged degradative enzyme substrate or neutral degradativeenzyme substrate. The separated positively charged degradative enzymeproduct is detected thereby detecting the degradative enzyme in thebodily fluid sample.

Within the bodily fluid sample is the degradative enzyme. Therefore, bycontacting the bodily fluid sample with a negatively charged degradativeenzyme substrate or neutral degradative enzyme substrate, thedegradative enzyme (within the bodily fluid sample) is contacted with anegatively charged degradative enzyme substrate or neutral degradativeenzyme substrate. Thus, in some embodiments, the degradative enzymewithin the bodily fluid sample is contacted with the bodily fluid samplewith a negatively charged degradative enzyme substrate or neutraldegradative enzyme substrate.

A degradative enzyme is an enzyme with the ability to degrade (e.g.hydrolyze) biological molecules. In some embodiments, the degradativeenzyme is a protease, a lipase, an amylase, or a nuclease. A protease isa degradative enzyme that cleaves peptide bonds linking amino acids in apolypeptide chain. A lipase is a degradative enzyme that cleaves esterbonds in lipid substrates. An amylase is a degradative enzyme thatcleaves glycosidic bonds in polysaccharides. A nuclease is degradativeenzyme that cleaves phosphodiester bonds in nucleic acids. In someembodiments, the degradative enzyme is a protease, such as a serineprotease, a threonine protease, a cysteine protease, an aspartic acidprotease, a metalloprotease or a glutamic acid protease. Degradativeenzymes also include, but are not limited to chymotrypsin, trypsin,matrix metalloproteases, and thrombin. In some embodiments, thedegradative enzyme is α-chymotrypsin, trypsin, elastase, matrixmetalloproteinase-2 (MMP-2), MMP-9, MMP-14, or both α-chymotrypsin andtrypsin.

Very low levels of degradative enzymes (e.g. chymotrypsin, trypsin,matrix metalloproteases, peptidases, thrombin, amylases, lipases,nucleases, kinases) may be detected directly in blood, plasma and otherbiological samples using the degradative enzyme substrates and othercharge differentiating substrates provided herein. The degradativeenzymes, in some embodiments, are the initiates and earlier indicatorsof shock, inflammation and many other disease processes.

A bodily fluid sample, as described herein, is a sample of fluidobtained from the body of a subject. Examples of a bodily fluid samplesinclude, but are not limited to blood, plasma, serum, urine, saliva,synovial fluid, lymph fluid, semen, intestinal fluid samples, fecalfluid samples, milk, biopsy and smear samples and other biological orenvironmental samples. A bodily fluid sample, as disclosed herein, mayinclude constituents that are detectable (e.g. fluorescent), chargedand/or capable of migrating on a typical electrophoresis gel apparatus.In some embodiments, the detectable constituents of the bodily fluidsample are fluorescent.

In other embodiments, the bodily fluid sample is a crude bodily fluidsample. A “crude bodily fluid sample” refers to a bodily fluid samplecontaining the endogenous constituents of the bodily fluid sample asfound in the subject and optionally additional assay components (e.g.buffers, stabilizing reagents, etc.). Thus, a crude bodily fluid sampleis not the product of substantial purification or separation procedures,such as size exclusion filtering or separation centrifugation. In otherembodiments, the bodily fluid sample is a crude blood sample or a crudelymphoid sample. In some embodiments, the bodily fluid sample is a crudeblood sample.

In some embodiments, the bodily fluid sample is a semi-crude processedbodily fluid sample. A “semi-crude bodily fluid sample” is a bodilyfluid sample containing at least 50% by weight of the endogenousconstituents of the bodily fluid sample as found in the subject andoptionally additional assay components.

In other embodiments, the bodily fluid sample is a processed interferingbodily fluid sample. A “processed interfering bodily fluid sample” is abodily fluid sample which has been processed (e.g. partially purified,separated, concentrated etc.) but retains endogenous constituents of thebodily fluid sample as found in the subject.

In other embodiments, the bodily fluid sample is a cell-free bodilyfluid sample. A “cell-free bodily fluid sample” is a bodily fluid samplefrom which all or some of the cellular constituents have beensubstantially removed. In some embodiments, the cell-free bodily fluidis blood plasma. In other embodiments, the bodily fluid sample is aprocessed cell-free bodily fluid sample. A “non-clotting blood sample”is a bodily fluid blood sample from which clotting factors have beensubstantially removed. In some embodiments, the non-clotting fluidsample is blood serum.

In some embodiments, the methods provided herein allow for rapiddetection of degradative enzymes. The bodily fluid sample obtained froma subject may be used directly in the methods disclosed herein. In somecases, some minimal sample preparation may be employed such as addingagents to the sample, such as buffers, activating agents, stabilizingagents, and the like.

A degradative enzyme substrate is a molecule (e.g. a biologicalmolecule) recognized and cleaved by a degradative enzyme. Upon cleavage,the degradative enzyme substrate is converted to a degradative enzymeproduct. Degradative enzyme substrates may include, but are not limitedto, peptides, lipids, polysaccharides and nucleic acids. In someembodiments the degradative enzyme substrate is a negatively chargeddegradative enzyme substrate or a neutral degradative enzyme substrate.In some embodiments, the negatively charged degradative enzyme substrateor neutral degradative enzyme substrate includes a peptide, a lipid, apolysaccharide or a nucleic acid. In other embodiments, the negativelycharged degradative enzyme substrate or neutral degradative enzymesubstrate is a peptide.

The degradative enzyme substrates disclosed herein are typicallydesigned to avoid unspecific cleavage by degradative enzymes present inthe bodily fluid sample or other analysis constituents. To increase thespecificity of degradative enzyme cleavage, an isomeric amino acid, anon-amino acid group, a stabilizing moiety or combinations thereof maybe included in the degradative enzyme substrate at specific positions.In some embodiments, the isomeric amino acid is a D-amino acid. In otherembodiments, the non-amino acid group is a polyethylene glycol. Theposition of the isomeric amino acid or non-amino acid group within thedegradative enzyme substrate may be critical. In some embodiments, theisomeric amino acid or non-amino acid group is introduced near thecleavage recognition site. The cleavage recognition site is a specificsequence of biological molecules that is recognized and cleaved by adegradative enzyme. Upon introduction of the isomeric amino acid ornon-amino acid group near the cleavage recognition site, cleavage byunspecific degradative enzymes is prevented. In some embodiments, theisomeric amino acid or non-amino acid group is positioned more than twoto three residues from the cleavage recognition site of the degradativeenzyme.

In some embodiments, the degradative enzyme substrate includes astabilizing moiety. In order to prevent unspecific degradation of thedegradative enzyme substrate, a stabilizing moiety may be included inthe degradative enzyme substrate. Unspecific enzyme degradation may becaused by the presence of degradative enzymes such as peptidases orunspecific proteases present in the bodily fluid sample or the assaycomponents. Examples of stabilizing moieties may include any appropriatechemical modification of an amino acid. Such chemical modifications mayinclude for example phosphorylation, ubiquitination, acetylation,amidation, glycosylation, covalent attachment of flavin,ADP-ribosylation, cross linking, iodination, methylation, and the like.

In other embodiments, the degradative enzyme substrate includes adetectable moiety. A detectable moiety is a moiety that confersdetectability to the degradative enzyme substrate using known methods inthe art. For example, the degradative enzyme substrate may includedetectable moieties such as organic fluorophores, detectable moietiesemployed in fluorescent energy transfer (FRET) systems, quantum dots,fluorescent nanoparticles, dendrimeric nanoparticle labels, gold andother metallic nanoparticles, carbon nanotubes, chemiluminescent labels,HRP, microperoxidase, alkaline phosphatase and electrochemical andoxidation/reduction labels for direct electronic detection. Examples forfluorophores include, but are not limited to, fluoresceinisothiocyanate, rhodamine, coumarin, cyanine, Alexa Fluors, DyLightFluors, luciferin, green fluorescent protein, fluorescein, Texas Red,Cy3, Cy5 and Bodipy dyes. Many such labels are commercially availablefrom, for example, the SIGMA chemical company (Saint Louis, Mo.),Molecular Probes (Eugene, Oreg.), R&D systems (Minneapolis, Minn.),Pharmacia LKB Biotechnology (Piscataway, N.J.), CLONTECH Laboratories,Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company(Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies,Inc. (Gaithersburg, Md.), Fluka Chemica-Biochemika Analytika (FlukaChemie AG, Buchs, Switzerland), and Applied Biosystems (Foster City,Calif.), as well as many other commercial sources known to one of skill.Furthermore, those of skill in the art will recognize how to select anappropriate fluorophore for a particular application and, if it is notreadily available commercially, will be able to synthesize the necessaryfluorophore de novo or synthetically modify commercially availablefluorescent compounds to arrive at the desired fluorescent label. Insome embodiments, the detectable moiety includes a positively chargedfluorophore. In certain embodiments, the detectable moiety is achemiluminescent moiety. In other embodiments, the detectable moiety isa fluorophore, a quantum dot, a fluorescent nanoparticle, a dendrimericnanoparticle, a metallic particle, a chemiluminescent label, anelectrochemical label or a oxidation/reduction label. In otherembodiments, the detectable moiety is a fluorophore. In addition toconferring detectability, the detectable moiety may also change the netcharge of the degradative enzyme substrate and the overall secondaryspecificity of the substrate for a specific enzyme. More, specifically,the fluorophore may be carefully considered as a modifying R-group forimproving secondary specificity of the substrate.

The degradative enzyme substrate may include a detectable moiety or astabilizing moiety. In some embodiments, the degradative enzymesubstrate includes a detectable moiety and a stabilizing moiety. In someembodiments, the stabilizing moiety is attached to one end of thedegradative enzyme substrate and the detectable moiety is attached tothe opposite end of the degradative enzyme substrate. In someembodiments, where the degradative enzyme substrate is a peptide, thestabilizing moiety is attached to the N-terminal end of the degradativeenzyme substrate and the detectable moiety is attached to the C-terminalend. In other embodiments, where the degradative enzyme substrate is apeptide, the detectable moiety is attached to the N-terminal end of thedegradative enzyme substrate. In some embodiments, where the degradativeenzyme substrate is a peptide, the stabilizing moiety is attached to theC-terminal end of the degradative enzyme substrate.

The detectable moiety may be covalently attached to the degradativeenzyme substrate using a reactive functional group, which can be locatedat any appropriate position. When the reactive group is attached to analkyl, or substituted alkyl chain tethered to an aryl nucleus, thereactive group may be located at a terminal position of an alkyl chain.Reactive groups and classes of reactions useful in practicing thepresent invention are generally those that are well known in the art ofbioconjugate chemistry. Currently favored classes of reactions availablewith reactive known reactive groups are those which proceed underrelatively mild conditions. These include, but are not limited tonucleophilic substitutions (e.g., reactions of amines and alcohols withacyl halides, active esters), electrophilic substitutions (e.g., enaminereactions) and additions to carbon-carbon and carbon-heteroatom multiplebonds (e.g., Michael reaction, Diels-Alder addition). These and otheruseful reactions are discussed in, for example, March, Advanced OrganicChemistry, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson,Bioconjugate Techniques, Academic Press, San Diego, 1996; and Feeney etal., Modification of Proteins; Advances in Chemistry Series, Vol. 198,American Chemical Society, Washington, D. C., 1982.

Linkers may also be employed to attach the detectable moiety to thedegradative enzyme substrate. Linkers may include reactive groups at thepoint of attachment to the detectable label and/or the mobile detectableanalyte binding reagents. Any appropriate linker may be used in thepresent invention, including substituted or unsubstituted alkylene,substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycoalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, andsubstituted or unsubstituted heteroarylene. Other useful linkers includethose having a polyester backbone (e.g. polyethylene glycol), nucleicacid backbones, amino acid backbones, and derivatives thereof. A widevariety of useful linkers are commercially available (e.g. polyethyleneglycol based linkers such as those available from Nektar, Inc. ofHuntsville, Ala.). The detectable moiety may also be non-covalentlyattached to the degradative enzyme substrate using any appropriatebinding pair (e.g. biotin-streptavidin, his tags, and the like).

Upon degradative enzyme cleavage the degradative enzyme substrate isconverted into a degradative enzyme product having a different netcharge than the degradative enzyme substrate. The generation of adegradative enzyme product having a different net charge than thedegradative enzyme substrate ensures efficient subsequent separation ofthe degradative enzyme product from the degradative enzyme substrate.Further, a positively charged degradative enzyme product can beseparated from detectable constituents of the bodily fluid samplethereby increasing the detection sensitivity. In some embodiments, adegradative enzyme reacts with a negatively charged degradative enzymesubstrate or neutral degradative enzyme substrate thereby forming apositively charged degradative enzyme product. In other embodiments, thepositively charged degradative enzyme product is separated fromdetectable constituents of the bodily fluid sample.

As described above, the degradative enzyme substrate may include adetectable moiety. Upon reaction with the degradative enzyme, thedetectable moiety becomes part of the positively charged degradativeenzyme product and confers all the properties previously described for adetectable moiety to the positively charged degradative enzyme product.With the inclusion of a detectable moiety, the positively chargeddegradative enzyme product becomes detectable and thereby thedegradative enzyme in the bodily fluid sample may be detected. In someembodiments, the negatively charged degradative enzyme substrate orneutral degradative enzyme substrate and the positively chargeddegradative enzyme product include a detectable moiety.

In some embodiments, the degradative enzyme substrate is attached to asolid support. Attachment of the degradative enzyme substrate isperformed such that upon cleavage of the degradative enzyme substrate bythe degradative enzyme the degradative enzyme product remains attachedto the solid support. Attaching the degradative enzyme product to asolid support allows separation of the degradative enzyme product fromthe bodily fluid sample and other constituents after cleavage with thedegradative enzyme. The choice of solid support for use in the presentmethods is based upon the desired assay format and performancecharacteristics. Acceptable solid supports for use in the presentmethods can vary widely. A solid support can be porous or nonporous. Itcan be continuous or non-continuous, and flexible or nonflexible. Asolid support can be made of a variety of materials including ceramic,glass, silicon, metal, organic polymeric materials, or combinationsthereof.

In order to detect a degradative enzyme in a bodily fluid sample thenegatively charged degradative enzyme substrate or neutral degradativeenzyme substrate is contacted, and in some cases incubated, with thebodily fluid sample for sufficient time to allow degradation of thesubstrate. In some embodiments, about 100 to about 1000 μl of the crudebodily fluid sample are contacted with the negatively chargeddegradative enzyme substrate or neutral degradative enzyme substrate. Inother embodiments, about 1 to about 1000 μl of the crude bodily fluidsample are contacted with the negatively charged degradative enzymesubstrate or neutral degradative enzyme substrate. In anotherembodiment, about 0.1 to about 10 μl of the crude bodily fluid sampleare contacted with the negatively charged degradative enzyme substrateor neutral degradative enzyme substrate.

Another component of the method is the separation of the degradativeenzyme product from the degradative enzyme substrate and detectableconstituents of the bodily fluid sample prior to detection. Thisseparation can be achieved through application of an electric field incombination with size exclusion. A specific net charge typically confersa differential migratory potential to the degradative enzyme product andthe degradative enzyme substrate when an electric field strength isapplied. Additionally, due to the difference in size between thedegradative enzyme substrate and the degradative enzyme product,separation is also accomplished through size exclusion. In someembodiments, the positively charged degradative enzyme product iselectrophoretically separated from the negatively charged degradativeenzyme substrate or neutral degradative enzyme substrate. In someembodiments, the process of electrophoretically separating is performedusing a gel electrophoresis. In another embodiment, the gelelectrophoresis is a gradient gel electrophoresis.

The concept of the present invention can easily be applied to newdiagnostic, monitoring and detection devices and systems which include,but are not limited to microtiter plate formats, focusing microgels(smart gels), lab-on-a-chip devices, micro/nanoarrays, microsensordevices and point of care (POC) systems which will utilize the uniquenet charge differentiating protease substrates (NCDPS). The methodsprovided herein may be designed to detect a variety of clinicallyimportant protease activities (and other markers) simultaneously in amultiplex format that include but are not limited to micro/nanoarrays,point of care systems and devices for critical care and emergency roomapplications.

In some embodiments, the devices and other charge changing substrateconstructs (antibodies, etc.) are designed to detect the actual cleavageproducts, e.g., amino acid sequences that are derived from importantelements of membrane receptors for physiological function (e.g. theextracellular domain of the insulin receptor, membrane adhesionreceptor, growth factor receptors, and many others membrane receptors),plasma proteins, or functional receptors in specific organs (nicotinicreceptor in the brain, amyloid protein in Alzheimers disease, glutamatereceptors, adrenergic receptors, cholinergic receptors, amino acidtransporters, selectins, glycocalyx proteins, and many others). Incertain embodiments, the combined properties of the unique substratesand devices allow the cleaved product fragments to be rapidly andclearly separated from the substrate, blood cells and background bloodand plasma components which can greatly interfere and limit thedetection of very low levels of disease specific enzymes. Suchanalytical, research and diagnostic devices/systems may include and/orincorporate without limitation, electrophoretic, dielectrophoretic,microfluiduic manipulation and/or differentiation components; on-chipoptical/fluorescent excitation (semiconductor lasers) and detection(APDs, CCDs, etc.) components; micro/nanoscale electrodes formanipulation and/or electrochemical, impedance, potentiometric andamperometric detection; and nanopore materials and components forseparation and selection.

FIGS. 4A-4C show the advantages achieved from the combined properties ofa unique substrate and device disclosed herein that allows cleavedproduct fragments to be rapidly and clearly separated from the originalsubstrate, blood cells and background blood and plasma components. FIG.4A first shows the negatively charged substrate mixed with blood withinthe sample well of the electrophoretic separation device, after beinggiven a short time to react with a target enzyme (usually about one tothirty minutes). Cleavage of the substrate by the specific enzymeproduces a positively charged fluorescent product fragment. In FIG. 4Bthe result of DC electric field application are shown. The positivelycharged fluorescent peptide cleavage fragment moves out from the samplewell into a low density gel towards the negative electrode (anode) andis rapidly separated from blood cells which remain in the sample well.The un-cleaved negatively charged substrate and other blood and plasmacomponents (mostly negatively charged proteins, hemoglobin, albuminetc.) migrate into the low density gel towards the positive electrode(cathode). The positively charged fluorescent peptide cleavage fragmentmoves quickly for a short distance where it reaches a high-densityfocusing gel. This focusing gel concentrates the product fragment intosharp narrow fluorescent band providing a higher signal to noise ratiofor greatly improved detection sensitivity. Depending on the devicedesign and sample well/chamber dimensions, the fluorescent peptideproduct fragment may only have to migrate less than one centimeter in alarge scale device; less than a millimeter in a miniaturized device; orless than 100 microns in a microscale device. The actual separation timerequired depending on sample size may be less than one minute. Dependingon the research or diagnostic application, devices can be designed forsample volumes that range from several milliliters to less than ananoliter.

A variety of unique devices and systems are disclosed which worksynergistically with the NCDPS substrates and methods of this inventionfor the rapid and highly sensitive detection of clinically relevantproteases and enzymes directly in blood, plasma and other clinical orbiological samples. Depending on the application, these devices includebut are not limited to microtiter plate formats, focusing gel/smart geland focusing microgels in various cartridge formats, microarray devicesin cartridge type formats. The devices can be used in association withpower supplies, a manual or fluidic system for sample application, afluorescent or other detection system and a computer based datacollection system. These devices (microtiter plate, focusing gel, etc.)can also part of fully integrated laboratory or clinical diagnosticsystem which contains automated sample handling and integrated fluidics,detection, power and data processing components. In most cases, thesample cartridge devices are designed to be relatively inexpensive anddisposal. In other aspects of the invention, smaller more compactdevices and systems can be designed which are commonly called point ofcare (POC) systems, fully integrated portable field systems and evenmore miniaturized lab-on-a-chip systems. In certain cases the devicesand systems of this invention have major advantages over more classicalsystems in that they eliminate or greatly reduce the need for any samplepreparation (true sample to answer systems).

FIG. 13 shows the design for just one type of microtiter plateseparation and detection system for multiple sample and/or multiplexdegradative enzyme analyses using NCDPS substrates. In this case, bloodor other samples mixed with the appropriate NCDPS (negatively chargedsubstrates) are placed in the sample wells of the microtiter plateseparation/detection device. After being given a short time to reactwith the target enzyme in the sample (generally one to thirty minutes),cleavage of the substrate by the target enzyme produces the positivelycharged fluorescent product fragments. When the DC electric field isapplied, the positively charged degradative enzyme product moves throughthe sample chamber toward the negative electrode (anode). The positivelycharged degradative enzyme products are rapidly separated from bloodcells, un-cleaved negatively charged substrate and other blood andplasma components (mostly negatively charged proteins, hemoglobin,albumin etc.) which migrate towards the positive electrode (cathode) onthe opposite side of the microtiter plate sample chamber.

In the case of this format, the electrodes are placed behind amicrotiter plate chamber wall which can be composed of a porous membrane(cellulose, nylon, plastic, etc.), a controlled pore filter material(glass, ceramic, etc) or an agarose/polyacrylamide gel composite whichseparates the electrodes from the actual sample chambers. The positivelycharged degradative enzyme product moves quickly for a short distancewhere it reaches the sample chamber wall. At this point, the microtiterplate walls can either be designed with a high pore density structuresuch that the fluorescent fragments concentrate onto the wall itself, orquickly migrate through the wall with low pore density structure andthen concentrate or focus on a high pore density gel or other materialfor enhanced detection. In most cases, the entire separation anddetection time after the sample reaction would be less than fiveminutes. Thus, the total sample to answer time for most applicationscould be less than thirty minutes. In most cases for this format, thefocused fluorescent bands can be scanned and detected by anepifluorescent or other suitable fluorescence/luminescence detectordevice.

Another type of device which this invention discloses are focusing geland smart gel formats which can be designed in large scale, miniaturescale and microscale forms. These formats are somewhat akin toelectrophoretic horizontal/submarine and vertical gel formats. Suchfocusing gel devices can also be designed in multiple tube and capillaryformats. A focusing gel normally concentrates the fluorescent band injust the z-dimension (makes a broad fluorescent band much narrower). Asmart gel is designed in a way that causes the fluorescent band toconcentrate in all three dimensions (X-Y-Z). More specifically, a broadfluorescent band would now be focused or concentrated into a mall pointof intense fluorescence. Smart gel formats involve the construction ofcone shape structure through which the fluorescent band migrates.

FIG. 14A shows the design for just one type of focusing gel system formultiple sample and/or multiplex enzyme analyses using NCDPS substrates.In this case, blood or other samples mixed with the appropriate NCDPS(negatively charged substrates) are placed in the sample wells of thedevice. After being given a short time to react with the target enzymein the sample (generally one to thirty minutes), cleavage of thesubstrate by the target enzyme produces the positively chargedfluorescent product fragments. When the DC electric field is applied,the positively charged degradative enzyme product moves from the samplechamber toward the negative electrode (anode). The positively chargeddegradative enzyme products are rapidly separated from blood cells,un-cleaved negatively charged substrate and other blood and plasmacomponents (mostly negatively charged proteins, hemoglobin, albuminetc.) which migrate towards the positive electrode (cathode). Thepositively charged degradative enzyme product moves quickly for a shortdistance into the low pore density gel and then reaches a high poredensity gel or other material which concentrates and focuses thefluorescent fragment for enhanced detection. In most cases, the entireseparation and detection time after the sample reaction would be lessthan five minutes. Thus, the total sample to answer times for mostapplications could be less than thirty minutes. In most cases for theseformats the focused fluorescent bands can be scanned and detected by anepifluorescent or other suitable fluorescence/luminescence detectordevice. In miniaturized or microscale versions of this device, detectionwould be carried out by an integrated detector system using wave guides,diode lasers and a solid state CMOS or avalanche photodiode detector.

In some embodiments, a non-fluorescent negatively charged polymericentity (polyglutamic acid, DNA, etc.) can be run from the oppositedirection of the focusing gel so that it meets the positively chargedfluorescent fragment, which becomes neutralized, non-mobile and morehighly focused. This technique further improves the gel focusing processand subsequent detection sensitivity. This same strategy can be used asa trigger mechanism for very sensitive chemiluminescent detection wherea positively charged peptide fragment with a chemiluminescent labelmeets a chemiluminescent catalytic molecule such as peroxidase or aluciferase. Another similar strategy can be used where a positivelycharged peptide fragment with an oxidation or reduction label is movedfrom the reaction chamber through a gel into an electrochemical sensoror detecting element. For the various devices described above,particularly if designed to be disposable, sample chambers into whichthe reagents (substrates) have been pre-filled or lyophilized will havea significant advantage of producing a one step assay. A sample (blood,plasma, etc.) is added to the device and the assay is run without theneed to add any reagents.

Other devices which represent just a few of the many possible devices ofthis invention are discussed below. FIG. 15 shows the concept for amicroelectrode array type smart gel device. FIG. 16 shows the conceptfor a microfluidic type smart gel device. FIG. 17 shows the concept fora protease reaction accelerator and detector device.

In some embodiments, the total time to detect protease activity in theinvention has two main considerations: (1) the time it takes for thereaction of the protease and net charge differentiating proteasesubstrate and (2) the time it takes for electrophoresis of thefluorescent cleavage products and the subsequent detection of thefluorescence. The latter can be performed rapidly (seconds to minutes)because of the “Smart Gel” technology and because of highly sensitivefluorescent detectors (charge coupled device (CCD) cameras,photomultiplier tubes (PMT)). The former, therefore, can be the ratelimiting step for this detection scheme if the reaction takes longerthen the electrophoresis/detection time.

In order to reduce the reaction time, there are several optionsavailable: (1) introduce more substrate into the detection sample inorder to improve reaction kinetics or (2) use an electric field toconcentrate the degradative enzyme and the degradative enzyme substratetogether to improve reaction kinetics. The latter method is cheaper asit does not require additional reagents, but instead concentrates thereagents already present in the detection sample. This works because thedegradative enzyme and the degradative enzyme substrate are bothnegatively charged and can be actively concentrated with an electricfield.

The device shown in FIG. 17 achieves this by first using an electricfield to concentrate the negatively charged substrate and protease intoone part of the device to accelerate the reaction kinetics, and thenuses the electric field to concentrate the positively chargedfluorescent product fragments into another part of the device fordetection. FIG. 18 shows another version of this concept, but in thiscase the fluorescent substrate is immobilized to the surface of thedevice to where the protease will be concentrated. Such devices withimmobilized substrates or with sample chambers where the reagents(substrates) have been lyophilized have a significant advantage in thatsamples can be applied and run without the need to add a reagent.

As described above methods are provided that, in some embodiments,rapidly separate a positively charged degradative enzyme from anegatively charged or neutral degradative enzyme substrate andconstituents of the bodily fluid sample. In those embodiments where thedegradative enzyme product includes a detectable moiety, the degradativeenzyme product may be readily detected subsequent to the separationprocess. Detection units such as epifluorescent detectors,electrochemical detectors or chemiluminescent detectors may be used inthe methods described herein. In some embodiments, detecting theseparated positively charged degradative enzyme product includesdetecting a fluorophore. In some embodiments, the fluorescent signal canbe readily resolved from background components by two mechanisms: (a)exclusion of background components larger then fluorescent productfragment by filtration (e.g. in an electrophoretic gel, blood cellscannot leave the sample loading well because they are too large tomigrate into the pores of the gel) and (b) exclusion of backgroundcomponents that are the same size as or smaller then the fluorescentproduct fragment because of opposite charge (e.g. in an electrophoreticgel, positively charged fluorescent product will migrate in the oppositedirection from negatively charged proteins, blood cell fragments, etc.).In another embodiment, detecting the separated positively chargeddegradative enzyme product includes detecting a chemiluminescent label.

The specific features of the degradative enzyme substrates have beendescribed herein. They may include a stabilizing moiety, a detectablemoiety, a particular net charge and additional features conferringspecificity for a particular degradative enzyme. The following tablediscloses certain useful specific peptide sequences. In someembodiments, the methods described herein include a negatively chargeddegradative enzyme substrate or neutral degradative enzyme substrateincluding one or more of the amino acid sequences (also referred toherein as peptide sequences) set forth as SEQ ID NOs:1-18 andconservative amino acid substitutions thereof.

In some embodiments, the negatively charged degradative enzyme substrateor neutral degradative enzyme substrate includes one or more of theamino acid sequence set forth as SEQ ID NOs:1-18 or conservative aminoacid substitutions thereof and a detectable moiety. In anotherembodiment, the detectable moiety is a fluorophore, a quantum dot, afluorescent nanoparticle, a dendrimeric nanoparticle, a metallicnanoparticle, a chemiluminescent label, a electrochemical label or aoxidation/reduction label. In another embodiment, the detectable moietyis a fluorophore. In one embodiment, the detectable moiety is achemiluminescent label.

In other embodiments, the negatively charged degradative enzymesubstrate or neutral degradative enzyme substrate includes one or moreof the amino acid sequence set forth as SEQ ID NOs:1-7 or conservativeamino acid substitutions thereof and a stabilizing moiety (as describedabove). In other embodiments, the negatively charged degradative enzymesubstrate or neutral degradative enzyme substrate includes one or moreof the amino acid sequence set forth as SEQ ID NOs:1-7 or conservativeamino acid substitutions thereof, a detectable moiety, and a stabilizingmoiety.

TABLE 1 SEQ ID NO: NCDPS Amino Acid Sequence Degradative Enzyme 1Gly-Asp-Ala-Gly-Tyr-/-Ala-Gly-Ala-Gly-Lys α-Chymotrypsin 2Asp-Gly-Asp-Ala-Gly-Arg-/-Ala-Gly-Ala-Gly-Lys Trypsin 3Asp-Ala-Gly-Ser-Val-Ala-Gly-Ala-Gly-Lys Elastase 4Gly-Asp-(Leu-Ala-Ala-/-Ile-Thr-Ala)-Ala-Gly-Ala-Gly-Lys MMP-2 5Gly-Asp-(Pro-Val-Gly-/-Leu-Thr)-Ala-Gly-Ala-Gly-Lys MMP-9 6Gly-Asp-(Leu-Ile-Ser-His-Ser-/-Ile)-Ala-Gly-Ala-Gly-Lys MMP-14 7Asp-Gly-Asp-Ala-Gly-Tyr-/-Ala-Gly-Leu-/-Arg-/-Gly-Ala-LysChymotrypsin, Trypsin 8Acetyl-N-Gly-Asp-Ala-Gly-Tyr-/-Ala-Gly-Ala-Gly-Lys(Bodipy TR)-NH2α-Chymotrypsin 9Acetyl-N-Asp-Gly-Asp-Ala-Gly-Arg-/-Ala-Gly-Ala-Gly-Lys(Bodipy TR)-Trypsin NH2 10Acetyl-N-Asp-Ala-Gly-Ser-Val-Ala-Gly-Ala-Gly-Lys(Bodipy TR)-NH2 Elastase11 Acetyl-N-Gly-Asp-(Leu-Ala-Ala-/-Ile-Thr-Ala)-Ala-Gly-Ala-Gly- MMP-2Lys(Bodipy TR)-NH2 12Acetyl-N-Gly-Asp-(Pro-Val-Gly-/-Leu-Thr)-Ala-Gly-Ala-Gly-Lys(BodipyMMP-9 TR)-NH2 13Acetyl-N-Gly-Asp-(Leu-Ile-Ser-Hi6s-Ser-/-Ile)-Ala-Gly-Ala-Gly-Lys(BodipyMMP-14 TR)-NH2 14Acetyl-N-Asp-Gly-Asp-Ala-Gly-Tyr-/-Ala-Gly-Leu-/-Arg-/-Gly-Ala-Chymotrypsin, Trypsin Lys(Bodipy TR)-NH2 15N-Ac-Asp-PEG-Gly-Tyr-/-Ala-Gly- PEG-Bodipy TR Chymotrypsin 16N-Ac-Asp-Asp-PEG-Gly- Arg-/-Ala-Gly-PEG-Bodipy TR Trypsin 17Acetyl-N-Gly-(D-Asp)-(D-Ala)--Gly-Tyr-/-Ala-Gly-(D-Ala)-Gly-(D-Chymotrypsin Lys)(Bodipy TR)-NH2 18Acetyl-N-(D-Asp)-Gly-(D-Asp)-(D-Ala)-Gly-Arg-/-Ala-Gly-(D-Ala)-Gly-(D-Trypsin Lys)(Bodipy TR)-NH2

The specific protease peptide substrates listed above represent just afew of the potential sequences which can be designed and synthesized.The sequences shown above are labeled via the ε-amino group of lysinewith a Bodipy TR fluorophore, and the terminal α-amino groups areacetylated and the terminal α-carboxyl groups are modified to form anamide group (thus the terminal ends of these particular peptidesubstrates are not charged). It should be pointed out that other designparameters which include end labeling with an appropriate fluorescentgroups, and maintaining charge on the terminal ε-amino group and/or theterminal α-carboxyl group can be used when they produce the separationand detection advantages discussed in this invention. For the abovesequences the basic design parameters include specific amino acidcleavage sequences (for specific proteases), and appropriate chemistryfor further derivatization with an appropriate fluorescent label. A widevariety of modification and derivatization chemistries are well known inthe art for attaching fluorescent and other detection moieties topeptides. But included herein are also other novel design parameterswhich reduce secondary cleavage by other proteases (which might be foundnormally in blood) and most importantly, produce a positively chargedcleavage fragment from a negatively charged or neutral substrate whencleaved by the specific protease.

Other types of novel Net Charge Differentiating Protease Substrates(NCDPS) that include a short (2-6 residue) amino acidrecognition/cleavage site surrounded by either D-amino acids ornon-amino acid groups (either on one side or both sides and that can becharged or uncharged) are hereby disclosed. Previously known peptidesubstrates (often fluorogenic or chromogenic) typically have limitedspecificity for their intended protease targets since they ignore aprotease's specificity on the c-terminal side of the scissile (cleavage)bond. Longer peptide substrates (e.g. NCDPS, fluorescent resonant energytransfer substrates) can be made to create more specific substrates thataccount for this missing sub-site specificity. They also may allow forconjugation of various types of fluorescent moieties at greaterdistances from the scissile bond in order to reduce steric hindrancefrom the fluorophore, which would reduce the substrate's ability toenter the protease's active site.

The introduction of D-amino acid and non-amino acid groups may also helpto limit the introduction secondary cleavage sites into the substrate.Additionally, it allows for the incorporation of other properties intothese substrates, such as special arrangements of the charged groups toeliminate non-specific binding, better solubility properties orselective steric hindrance which would allow substrate to be moreefficiently cleaved by its specific protease, but not by a closelyrelated protease which has some specificity for the same cleavage site.However, as these substrates become longer, there is a risk ofincreasing the amount of non-specific proteolytic cleavage. As thenumber of L-amino acids in the substrate increases, there arepotentially more cleavage sites for proteases that were not intended tocleave that substrate. Thus, in order to obtain a substrate long enoughto a) allow for optimal spacing between the scissile bond and thefluorophore, b) include desired charge and solubility properties, and c)and include c-terminal sub-site specificity without increasingnon-specific cleavage, new substrates can be made that incorporateD-amino acids and non-amino acid groups into the substrate. They willprovide the desired properties of the substrate while allowing thespecificity of the substrate to be maintained or improved. The use ofD-amino acids represents just one example of designing more specificNCDPS substrates For example,Acetyl-N-Gly-Asp-Ala-Gly-Tyr-/-Ala-Gly-Ala-Gly-Lys(Bodipy TR)-NH₂ (SEQID NO:8) andAcetyl-N-Asp-Gly-Asp-Ala-Gly-Arg-/-Ala-Gly-Ala-Gly-Lys(Bodipy TR)-NH₂(SEQ ID NO:9) are a chymotrypsin and trypsin NCDPS, respectively. Theyare cleaved on the C-terminal side of the tyrosine and arginineresidues, respectively. Many of the residues in those substrates mayserve primarily to introduce sufficient space between the scissile bondand the fluorescent moiety and to give the substrate the desired chargechanging properties when it is cleaved. It is possible that non-specificproteases such as elastase and proteinase K can still cleave thissubstrate (albeit with relatively low activities compared tochymotrypsin and trypsin). Examples of substrates that take advantage ofnon-amino acids would be the following:

Chymotrypsin Substrate: (SEQ ID NO: 26)N-Ac-Asp-PEG-Gly-Tyr-/-Ala-Gly- PEG-Bodipy TR Trypsin Substrate:(SEQ ID NO: 27) N-Ac-Asp-Asp-PEG-Gly- Arg-/-Ala-Gly-PEG-Bodipy TR Note:PEG = Polyethylene Glycol, and “-/-” denotes the scissile bondSimilarly, additional substrates can be obtained by incorporatingD-amino acids as follows:

Chymotrypsin Substrate: Acetyl-N-Gly-(D-Asp)-(D-Ala)--Gly-Tyr-/-Ala-Gly-(D-Ala)-Gly-(D-Lys)(Bodipy TR)-NH2 Trypsin Substrate:Acetyl-N-(D-Asp)-Gly-(D-Asp)-(D-Ala)-Gly-Arg-/-Ala-Gly-(D-Ala)-Gly-(D-Lys)(Bodipy TR)-NH2 Note: Bodipy TR is conjugatedto the epsilon-amino of the Lysine and these peptides′ c-termini areamidated.

In addition to substrates with small organic fluorophore, luminescentmolecule (luminol, etc.) and oxidation/reduction molecule labels, themethods include the use of larger fluorescent, luminescent, andchemiluminescent macromolecules and nano-entities, which can include butare not limited to fluorescent nanoparticles, quantum dots, metallicnanoparticles and nanorods (gold, silver, platinum, semiconductormaterials, etc), dendrimers, fluorescent proteins (phycobiliproteins,fluorescent antibodies, fluorescent streptavidin, etc), bioluminescentproteins (peroxidase, alkaline phosphatase, luciferase, calmodulin,etc.) and oxidation/reduction proteins and nanoparticles.

FIG. 7 shows a scheme for the design of an NCDPS chymotrypsin specificsubstrate with a fluorescent streptavidin nanoparticle label. Thisparticular design allows a biotinylated peptide sequence to be attachedto the nanoparticle via the biotin-streptavidin ligand binding. However,the peptide sequences can also be attached to the nanoparticlescovalently. The fluorescent nanoparticle peptide substrate is designedsuch that it has net negative or neutral charge until a specificprotease cleaves the peptide producing a fluorescent nanoparticleproduct with a net positive charge. In some embodiments, this inventionrelates to nanoparticles, macromolecules (proteins, dendrimers, etc.),modified polymers and biopolymers, as well as surface materials (glass,plastic, silicon, gold etc.) being used for the attachment of adegradative enzyme substrate molecule. In this case, a non-fluorescentnanoparticle (macromolecule, dendrimer, polymer, surface material) wouldbe derivatized with a degradative enzyme substrate, which upon cleavageby a specific protease or other enzyme releases a fluorescent peptidefragment which then can be readily separated from the nanoparticles andother components of the sample.

Other novel more three dimensional Net Charge Differentiating ProteaseSubstrates (NCDPS) with improved specificity due to more tertiarystructure that better mimics the protease's natural biologicalsubstrates (a protein molecule) are hereby also disclosed in thisinvention. Such substrates can improve the ability to distinguishbetween similar proteases that recognize similar amino acid sequences,such as to distinguish matrix-metalloproteases (MMPs) of which there are28 known so far, and to distinguish trypsin-like proteases (trypsin,kallikrein, plasmin, and thrombin) from each other. Biologicalsubstrates for proteases are generally 3-dimensional proteins which inaddition to the specific amino acid cleavage sites have conformationaround that sequence site to which a specific protease must be able tofit in order for the protease's active site to catalyze the cleavage ofthe specific sequence (see FIG. 8). More tertiary or three-dimensionalstructures can be mimicked in a number of ways, such as by incorporatingprolines to introduce kinks in the peptide and by strategicallyintroducing amino acids or other entities with bulkier side chains ofvarious sizes. These modifications would be done to better aid asubstrate to fit more specifically into the target protease's activesite and less well into the active site of a closely related protease.Because the three dimensional structures around many of the true naturalsubstrates are not known, these more three dimensional substratestructures would have to be initially determined by a combinatorialprocess, which might require the use of complex peptide arrays. However,this method includes the use of a small library of 10 to 12 peptidesequences in which one or two prolines and several bulkier D-amino acidsor L-amino acids such as tryptophan, phenyalanine, isoleucine,glutamine, arginine are randomly arranged about three to six amino acidaway from the cleavage site. The small libraries would thus test avariety of three dimensional projections around the basic cleavage site.The first small library of peptides would be tested against three orfour closely related proteases (MMP's for example) to determine whichhas the most specificity. Once a unique peptide substrate is determined,a second library group of 10 to 12 peptides would be designed withfurther modification around the particular proline/bulky amino acidsequence which is imparting the higher specificity. A uniquely specificpeptide substrate should be determined from this second library group ofpeptide substrates.

Net charge differentiating substrates for detection of non-proteasedegradative enzymes directly in blood, plasma and other biologicalsamples are also hereby provided. Like NCDPS, these fluorescentsubstrates have a net negative or neutral charge prior to cleavage andsubsequently produce fluorescently labeled positive-charged cleavageproducts after cleavage. This specific charge change, for reasonsdescribed above, can facilitate in-situ detection in bodily fluids andeliminates the need for sample preparation. To obtain Net ChargeDifferentiating Amylase Substrates (NCDAS), Net Charge DifferentiatingLipid Substrates (NCDLS), and Net Charge Differentiating NucleaseSubstrates (NCDNS), and net Charge Differentiating Protein KinaseSubstrates (NCDPKS) compounds would be designed and synthesized with thefollowing chemical moieties: a negative charge moiety (with charge Qn),a cleavable substrate moiety (with charge Qs), a positive charge moiety(with charge Qp), and a fluorescent tag (with charge Qf). To obtain asubstrate whose net charge is negative prior to cleavage and positiveafter cleavage, sufficient charge needs to be incorporated in thepositive charge and negative charge moieties. For example, if Qf and Qsare zero, then |Qn|>|Qp| would result in the correct net charge change.The generic structures for NCDAS, NCDLS, NCDNS are described below. Foramylases, which generally cleave α-1,4 glycosidic bonds, the substratewould have a basic polysaccharide structure with from two to six glucoseunits to which a positively charge fluorescent group is attached at oneend of the structure and a terminal carboxyl group is attached at theother end of the structure producing a neutral substrate. Upon cleavageof the α-1,4 glycosidic bond, a positively charged fluorescent productfragment is produced (see FIG. 9). For lipases, which generally cleaveester bonds in triacylglycerol type structures, the substrates wouldtypically have a basic triacylglcerol structure with one of the fattyacid chains modified with a positively charged fluorophore and one ofthe glycerol groups modified with a phosphate group producing an overallnegatively charged substrate. Upon cleavage of the fatty acid esterbonds a positively charged fluorescent product fragment is produced (seeFIG. 10). For nucleases, which generally cleave phosphodiester bonds inpolynucleotide structures, the substrate would typically have a basicpolynucleotide structures with one of the terminal nucleotides modifiedwith a positively charged fluorophore. Upon cleavage of thephophodiester bonds, a positively charged fluorescent nucleosidefragment is produced (see FIG. 11).

In some embodiments, specific detection of the degradative enzyme isperformed. Where the degradative enzyme is specifically detected, thedegradative enzyme is detected at a level at least about 2, 3, 4, 5, 10,10, 30, 40, 50, 100, or 1000 times higher than the level of detection ofall other degradative enzymes in the bodily fluid sample. Thus, in someembodiments, the methods provided herein enable specific detection ofproteases, lipases, amylases, and nucleases. The specific detection ofparticular degradative enzymes allows for specific detection ofparticular disease states as disclosed below.

The NCDPS's and other charge differentiating substrates of thisinvention may be designed to detect low levels of specific proteolyticenzymes (chymotrypsin, trypsin, matrix metalloproteases, peptidases,thrombin, etc) as well as other enzymes (amylases, lipases, nucleases,kinases etc.) directly in blood, plasma and other biological samples. Insome embodiments, the differential in charge between the negativelycharged degradative enzyme substrate or neutral degradative enzymesubstrate and the positively charged degradative product results in a 10to 100 fold increase in detection sensitivity.

This invention further discloses Multiplex Protease Detection Formats,even where only one particular protease is actually required fordiagnostics. Most protease detection systems examine the activity of asingle protease, but, as the following discussion will show, a multiplexdetection scheme is useful where protease detection is applied todisease diagnostics. A variety of peptide substrates can be designed forinflammatory response or other diseases. Each of the specific enzymesubstrate sequences carries a cleavage site at defined location topromote specific detection. Each of the different enzyme substrates islabeled with a different fluorophore (different emission maximum) whichallows the simultaneous detection of multiple enzymes (see FIG. 5 andFIG. 6). The cleavage of the peptide substrates will result in a changein the net charge on the complex and the cleaved products can then beseparated from the intact peptide substrate by application of a directedelectrophoretic field. Subsequent detection is performed with highsensitivity fluorescent detection device which detects the specificfluorescent signals at their different fluorescent emission wavelengths.

In another embodiment, to obtain improved specificity for proteasedetection, a method is provided to utilize multiple protease substratespreferentially cleaved by different proteases, to more accuratelymeasure the activity of a single target protease. In some embodiments,where the peptide substrates are non-specifically cleaved by otherproteases, a protease may be reacted with multiple substrates therebyproviding a unique signature of cleavage activity for that group ofsubstrates. This signature distinguishes proteases involved in thecleavage. For example, using only a single chymotrypsin substrate, suchas Acetyl-N-Gly-Asp-Ala-Gly-Tyr-/-Ala-Gly-Ala-Gly-Lys(BodipyTR)-NH₂ (SEQID NO:8), to detect chymotrypsin (which cleaves on the c-terminal sideof Tyr, Phe, Trp, and Met) may not account for non-specific cleavage ofthe substrate by a non-chymotrypsin protease such as elastase (whichcleaves on the c-terminal side of Ala, Gly, Val). Using, in addition tothe first substrate, a second substrate more specific to elastase, suchas Acetyl-N-Asp-Gly-Ala-Val-Gly-Ala-Val-Lys (Bodipy TR)-NH₂ (SEQ IDNO:28), it could be concluded that the first cleavage of the substratemay be at least partially attributed to elastase.

In some specific embodiments, the methods provided herein involve thedesign and synthesis of unique fluorescent/nanoparticle net chargedifferentiating peptide substrates (NCDPS) (embodiments of thenegatively charged degradative enzyme substrate or neutral degradativeenzyme substrate) for the highly sensitive and selective detection ofthe enzymes (proteases, matrix metalloproteases, lipases, amylases)associated with inflammatory cascade in blood or plasma.

Additionally, certain devices disclosed herein allow the productfragments to be rapidly and clearly separated from the blood/plasmacomponents which limit/interfere with detection are also provided. Morespecifically, substrates which upon cleavage by a specific enzyme,produce cleavage products which have an overall net charge that isdifferent from the original peptide substrate. By way of example, adegradative enzyme substrate with a (0 or −) net charge and a firstpeptide product with a negative (−) net charge and second fluorescentpeptide product with a positive (+) net charge can be rapidly separatedby application of an electric field, and then subsequently detected.

Other embodiments include the development of prototype fluorescentdetection systems and ultimately the development of novel diagnosticsystems in microtiter plate formats, microarray formats and lab-ona-chip formats for point of care (POC) applications. FIGS. 3A-3C, 4A-4C,5, 6, 7, 8, 9, 10, and 11 show the design and synthesis of certainfluorescent/nanoparticle net charge differentiating peptide substrates(NCDPS), as well as possible mechanism of NCDPS cleavage, separation anddetection in multiplex formats and some other types of NCDPS substrates.The sequence of one NCDPS substrate which was designed and synthesizedwasAc-N-Asp-Gly-Asp-Ala-Gly-Tyr-X-Ala-Gly-Leu-X-Arg-Gly-Ala-Gly-diamino-ethyl-BodipyFL (SEQ ID NO:29). FIG. 3A shows this substrate labeled with a greenfluorophore and having a net charge of −1. After reaction withchymotrypsin, two cleavage products are formed. The fluorescentlylabeled cleavage product has a net charge of +2. FIG. 3B shows aseparation scheme without an electric field being applied, where theun-cleaved substrate and cleaved product are unresolved. FIG. 3C showsseparation after applying the electric field, where the un-cleavednegatively charged substrate and fluorescently labeled positivelycharged cleavage product migrate in opposite directions. Thefluorescently labeled cleavage product can subsequently be detected withminimal interferences using a sensitive fluorescent detector. Thissubstrate is specific for chymotrypsin and trypsin, and the productfragments have been separated both in whole blood and plasma usingagarose gel and/or polyacrylamide gel electrophoresis formats. Thedetection sensitivity for α-Chymotrypsin in buffer using this substratewas less than 20 mU/mL or <0.3 μg/ml (see Example 2). The detectionsensitivity for α-Chymotrypsin in human plasma using this substrate wasless than 20 mU/mL or <0.3 μg/ml (see Example 3). The detectionsensitivity for α-Chymotrypsin substrate product fragments in rat bloodwas about 20 mU/mL or ˜0.3 μg/ml (see Example 4).

In some embodiments, several novel Fluorescent Net ChargeDifferentiating Protease Substrates (NCDPS) are provided for thedetection of proteases directly in bodily fluid samples including butnot limited to blood, plasma, serum, urine, saliva, lymph, semen milk,intestinal and fecal fluids, cerebral spinal fluid, smears and biopsiedspecimens are hereby disclosed. They are substrates that are specificfor α-chymotrypsin, trypsin, elastase, matrix metalloproteinase-2(MMP-2), MMP-9, MMP-14, and both α-chymotrypsin and trypsin. Prior tocleavage, these fluorescent peptide conjugates are negatively charged.After cleavage by specific proteases, the fluorescently labeled cleavageproducts are positively charged. Under an electric field, the un-cleaveddegradative enzyme substrates and the fluorescently labeled cleavageproducts will migrate in opposite directions. The fluorescently labeledcleavage products can subsequently be detected by a sensitivefluorescent detector (photomultiplier tube, CCD camera, etc.). Thus, incertain embodiments, these substrates have unique net charge change,from negative to positive upon cleavage, thereby facilitating directdetection of target proteases directly in bodily fluids such as bloodand plasma.

Under some conditions it is possible that a particular NCDPS molecule iscleaved non-specifically, and then that molecule can no longer be usedto detect the specific protease it was designed for. This may in somecases be an issue for detection of certain proteases in blood, wherethere is an unusually high level of an endogenous protease activitywhich interferes with target protease of interest (for example, a hightrypsin-like protease background would interfere with trypsin detectionif the substrate were susceptible to both enzymes). In order to improvethe specificity of detection of a given protease, and facilitatedetection in complex bodily fluids such as blood, two methods can usedto overcome the problem.

First, the peptide sequence can be improved in order to reducenon-specific cleavage. This may not be as effective, however, foreliminating non-specific cleavage by very non-specific proteases such aselastase. The solution, therefore, is to utilize specific inhibitorstogether with specific substrates in order to further improve thespecificity. These specific inhibitors could reduce non-specificcleavage and facilitate more specific detection directly in blood. Forexample, to detect chymotrypsin activity specifically, non-specificcleavage by other proteases would need to be greatly reduced if thesubstrate is susceptible to cleavage by those other proteases.Acetyl-N-Gly-Asp-Ala-Gly-Tyr-/-Ala-Gly-Ala-Gly-Lys (Bodipy TR)-NH2 (SEQID NO:8) is a NCDPS for chymotrypsin and would be cleaved on thec-terminal side of Tyr. It potentially can be cleaved by elastase aswell since it has several Ala and Gly residues and elastase can cleaveon the c-terminal side of Ala and Gly. Thus, in order to improvedetection for chymotrypsin in a sample, that sample would be pre-treatedwith a specific elastase inhibitor such as GlaxoSmithKline's compoundGW311616A. This represents just one example of how a protease inhibitorcould be use to improve detection of a specific protease.

IV. Methods of Detecting Bodily Fluid Biomolecules Using Antibody orNucleic Acid Probes

In another aspect, a method is provided for detecting a biomolecule in abodily fluid sample. The method includes contacting a bodily fluidsample with a first detection antibody and a second positively chargedantibody to form a detectable positively charged biomolecule conjugate.The detectable positively charged biomolecule conjugate iselectrophoretically separated from negatively charged endogenousmaterial present in the bodily fluid sample. The detectable positivelycharged biomolecule conjugate is then detected. The detection of thedetectable positively charged biomolecule conjugate is facilitatedthrough detection of the first detection antibody. Biomolecules include,but are not limited to, enzymes, proenzymes, proteins, antibodies,peptides, proteins, protein cleavage sites (from other proteases),peptide cleavage fragments, peptides, hormones, virus, smallbiomolecules and drug molecules.

Each antibody typically recognizes a distinct epitope site on thebiomolecule. In some embodiments, the first detection antibody includesan antibody that is conjugated at the Fc region to a fluorophore orfluorescent protein, polymer or a dendrimer that is sufficiently anionicsuch that the overall net charge of the antibody-conjugate will benegatively charged or neutral, even when the first detection antibody isbound to its antigen. The second positively charges antibody may includean antibody conjugated at the Fc region to a non-fluorescent polymer,dendrimer or other entities that are sufficiently cationic such that theoverall net charge of the detectable positively charged biomoleculesconjugate will be positively charged. In some embodiments, the positivecharge on the second positively charged antibody exceeds the amount ofnegative charge on the first detection antibody. After incubation offirst detection antibody and the second positively charged antibody withthe bodily fluid sample (e.g. a clinical sample containing thebiomolecule (or antigen) of interest (e.g. peptide fragment, protein,antibody, disease biomarker)), only biomolecules bound to both the firstdetection antibody and the second positively charged antibody willmigrate toward the cathode under an electric field.

In some embodiments, as disclosed above, the bodily fluid sample is ablood sample or a blood plasma sample. Components in a blood sample orplasma sample that are negatively charged (e.g. cells, proteins, DNA)will migrate toward the anode. In some embodiments where anelectrophoretic gel is employed, components that are much larger thanthe detectable positively charged biomolecules conjugate (such as cells)do not escape out of the sample loading wells of the gel since they aretoo large to migrate into the pores of the gel. Thus, in someembodiments using an electrophoretic gel, the fluorescent signal fromthe detectable positively charged biomolecules conjugate is resolvedfrom many of the background contributors within a bodily fluid sample,which would either remain in the well of the gel or migrate in theopposite direction (see FIG. 12).

As discussed in the background section, recent evidence has shown thatone form of insulin resistance may be caused by the proteolytic cleavageof the extracellular α-subunit of the insulin receptor by matrixmetalloproteinases (MMPs) [22]. This would cause the release of peptidecleavage fragments in the blood stream. The matrix metalloproteinaseMMP-9 cleaves the α-1 insulin receptor site between a glycine andtyrosine residue (-Pro-Glu-Cys-Pro-Ser-Gly↑Tyr-Thr-Met-Asn-Ser-Ser-)(SEQ ID NO:30). This cleavage would release unique peptide fragmentsinto the blood which could be important diabetes biomarkers. Thus, insome embodiments, specific antibodies to α-1 insulin receptor cleavagefragments are designed according to the methods provided herein for useas a diagnostic for diabetes and associated inflammation (see Example9).

The analog of this process for detecting DNA and mRNA is performed withtwo nucleic acid probes. The nucleic acid probes may independently beDNA or RNA probes complementary to two sequences of the target DNA ormRNA. Thus, in another aspect, a method is provided for detecting anucleic acid in a bodily fluid sample. The method includes contacting abodily fluid sample with a first detection nucleic acid and a secondpositively charged nucleic acid to form a detectable positively chargednucleic acid conjugate. The detectable positively charged nucleic acidconjugate is electrophoretically separated from negatively chargedendogenous material present (e.g. endogenous nucleic acid) in the bodilyfluid sample. The detectable positively charged nucleic acid conjugateis the detected. The detection of the detectable positively chargednucleic acid conjugate is facilitated through detection of the firstdetection antibody.

In some embodiments, the first nucleic acid probe includes a detectablemoiety (e.g. covalently or ionically bonded to the nucleic portion) thatis negatively charged due to its phosphodiester backbone. In certainembodiments, the second positively charged nucleic acid includes anucleic acid portion that is conjugated (e.g. covalently or ionicallybonded) to a cationic polymer with enough positive charge such that theresulting detectable positively charged nucleic acid conjugate ispositively charged. As with the antibody assay described above, thenucleic acid method may be resolved from background contributors in abodily fluid in an electrophoretic gel.

V. Methods of Diagnosing Disease States

The unique devices, substrates and methods of this invention may bedesigned to provide both rapid and highly sensitive detection ofclinically relevant proteases, lipases, amylases and other shock andinflammatory markers directly in blood and plasma. The ability to detectand monitor these important disease biomarkers directly in blood orplasma provides a number of important advantages. First, samplepreparation is costly, time consuming and adds complexity to the overalldiagnostic process. Second, sample processing can cause degradation andsignificant loss of the analyte (specific enzyme), thus reducing theoverall sensitivity of the assay. Third, sample processing can add moreinterfering substances to the final sample, i.e. rupture of blood cellscan introduce more non-specific enzymes into the final sample. Fourth,because of sample degradation that occurs when the blood is stored forany length of time before processing, rapid and direct analysis in freshblood may be important even if the diagnostic result is not neededimmediately.

In some embodiments, the disease produces an inflammatory response thatis detected using the methods provided herein. In certain embodiments,the inflammatory response and related disease diagnostics is a rapid,quantitative detection for key enzymes (chymotrypsin, trypsin, MMPs,lipases and amylases) in multiplex formats that utilize minimal samplesize and with sensitivity sufficient to detect baseline values incontrol blood or plasma. For chymotrypsin and other proteases, the levelof sensitivity may be about 0.1 to 1 IU/mL (<1 μg/ml, pmole-fmole/ml).Substrates and devices that may allow the product fragments to berapidly and clearly separated from the blood or plasma components, whichgreatly limit and interfere with the detection of the disclosedsubstrates. Since these enzymes are the initiates and earlier indicatorsof shock, inflammation and many other disease processes their detectionis important for early warning of shock and monitoring of inflammatorycascades; as well as for diagnosis of chronic inflammatory processesrelated to diabetes, hypertension, cancer and other diseases.

A. Diagnosis Examples (Statistics)

Protease activities in patient blood or plasma can be modeled by aGaussian random variable (see FIG. 1A and FIG. 1B). When using thesebiomarkers for disease diagnosis, an activity threshold must be set todifferentiate between the disease and healthy states. When the activitythreshold is lowered, a larger range of protease levels or activitiesare used to diagnose a patient with the disease and the diagnosticsensitivity increases. However, this comes at a cost because there is atrade-off between sensitivity and specificity. As the sensitivityincreases, the specificity decreases and more false positives occur.This trade-off becomes quite significant when distinguishing highlyoverlapping populations. For example, in various studies to determine ifMMP-2 and MMP-9 were significantly elevated in patients with type2-diabetes, there was statistically significant elevation of one or bothof those proteases compared to healthy patients (P<0.05) [23, 24], butthere was also strong overlap in the distributions of MMP-2,9 levels inhealthy and diabetic populations. If diabetes were to be diagnosed onMMP-2 or MMP-9 levels alone, the detection would be one of thefollowing: highly specific with no sensitivity, highly sensitive with nospecificity, or some compromise in between consisting of poorspecificity and poor sensitivity. In order to resolve these stronglyoverlapping populations, more independent variables such as theactivities of additional proteases, are needed to resolve the diseasedand healthy populations.

To briefly illustrate this point mathematically, Bayesian detectiontheory will be employed. Protease detection-based disease diagnosis canbe defined as a classification, such that, for a multidimensional vectory of measured information (e.g. activities of multiple proteases), thereexists an optimal partition of space

^(n)={y:m(y)=0}∪{y:m(y)=1}, where the probability of false positives PFis minimized, and the probability of detection PD is maximized [25]:

${m\left( \underset{\_}{y} \right)} = \begin{Bmatrix}1 & {\frac{p\left( \underset{\_}{y} \middle| H_{1} \right)}{p\left( \underset{\_}{y} \middle| H_{0} \right)} \geq \frac{p_{0}\left( {C_{10} - C_{00}} \right)}{p_{1}\left( {C_{01} - C_{11}} \right)}} \\0 & {\frac{p\left( \underset{\_}{y} \middle| H_{1} \right)}{p\left( \underset{\_}{y} \middle| H_{0} \right)} < \frac{p_{0}\left( {C_{10} - C_{00}} \right)}{p_{1}\left( {C_{01} - C_{11}} \right)}}\end{Bmatrix}$C₁₀ represents the cost of choosing hypothesis H₁ (e.g. patient hasdiabetes) when the true state was H₀ (e.g. patient does not havediabetes), etc. The a priori probabilities p0, p1 are perhapsunavailable for general cases, but can be estimated for certain cases.The costs may also be adjusted towards the particular clinicalsituation, where a false positive may be less injurious than a falsenegative. Now, for a set of independent variables, the false detectionprobabilities become the product of the conditional probabilities ineach dimension. Therefore, if a diagnosis is based on more independentvariables, there will be a higher likelihood of proper diagnosis (seeFIG. 2). If independent variables are unavailable, then minimallycorrelated dimensions would be preferable. By monitoring the activitiesof multiple proteases in the blood, multiplex detection schemes maintainboth high sensitivity and specificity.

VI. Kits

In another aspect, kits are provided that typically provide a convenientmeans for supplying necessary reagents for the methods described above,such as ancillary reagents, apparatuses, instructions and/or othercomponents necessary to implement the invention.

For example, in one embodiment, a kit for detecting a degradative enzymein a bodily fluid sample (e.g. crude bodily fluid sample) is provided.The kit includes a negatively charged degradative enzyme substrate orneutral degradative enzyme substrate. In some embodiments, the kitincludes an electrophoretic separation apparatus. In some embodiments,the electrophoretic separation apparatus is a gradient gel. In otherembodiments, the kit further includes a detector. In some embodiments,the detector is an epifluorescence detector. In other embodiments, thedegradative enzyme is a protease, a lipase, an amylase or a nuclease. Inone embodiment, the degradative enzyme is a protease. In someembodiments, the negatively charged degradative enzyme substrate orneutral degradative enzyme substrate is a peptide, a lipid, apolysaccharide or a nucleic acid. In some embodiments, the negativelycharged degradative enzyme substrate or neutral degradative enzymesubstrate is a peptide. In another embodiment, the crude bodily fluidsample is a crude blood sample or a crude lymphoid fluid sample. In someembodiments, the crude bodily fluid sample is a crude blood sample.Similar kits may also be provided for the methods of detectingbiomolecules and diagnosing diseases described above.

Other materials useful in the performance of the assays can also beincluded in the kit, including test tubes, transfer pipettes, and thelike. The kit may also include written instructions for the use of oneor more of the reagents described herein. The invention contemplatesadditional kits packaged to deliver, instruct and otherwise aid thepractitioner in the use of the invention. These additional kits includethose for the use of diagnostic embodiments of the invention, and theirconstruction is well known by those of skill in the art provided withthe reagents set forth herein.

The characteristics of the components of the methods described above areequally applicable to the kits provided herein.

VII. Examples

A major advantage in some embodiments of the methods provided herein isthe novel protease substrates (NCDPS) which can be used directly inblood or plasma. This is demonstrated in the following experimentalexamples.

FIGS. 1A-28 disclose, inter alia, the design and synthesis of certainunique fluorescent net charge differentiating peptide substrates(NCDPS); certain embodiments of NCDPS cleavage, separation anddetection; the design of certain embodiments of detection/separationdevices and systems for using NCDPS substrates bioanalytical anddiagnostic assays; and experimental results using certain NCDPSsubstrates.

Example 1—Electrophoretic Mobility of Streptavidin QuantumDot—Biotinyl-Gln-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 Derivative

This represents an initial experiment where a biotinylated peptide(Biotin-Gln-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, SEQ ID NO:24) wasattached to red fluorescent streptavidin quantum dots and the relativechanges in electrophoretic mobility were observed. The overall resultsdemonstrated a significant charge reduction (less negative) for Q-dotswhich have been derivatized with the peptide (see FIG. 19). Sample 1(0:1 Subst:Qdot) Control=3 uL Qdot 565 40 nM/0.5×TBE+3 uL 80 mM Tris HCLpH 7.8. Sample 2 (1:1 Subst:Qdot)=3 uL Qdot 565 40 nM/0.5×TBE+3 uLpeptide 40 nM/80 mM Tris HCL pH 7.8. Sample 3 (10:1 Subst:Qdot)=3 uLQdot 565 40 nM/0.5×TBE+3 uL peptide 4 00 nM/80 mM Tris HCL pH 7.8.Sample 4 (100:1 Subst:Qdot)=3 uL Qdot 565 40 nM/0.5×TBE+3 uL peptide 4uM/80 mM Tris HCL pH 7.8. Sample 5 (1000:1 Subst:Qdot)=3 uL Qdot 565 40nM/0.5×TBE+3 uL peptide 40 uM/80 mM Tris HCL pH 7.8. Sample 6 (10000:1Subst:Qdot)=3 uL Qdot 565 40 nM/0.5×TBE+3 uL peptide 400 uM/80 mM TrisHCL pH 7.8. Again, the overall results show that a positively chargedpeptide sequences can be designed which markedly influences theelectrophoretic mobility of fluorescent nanoparticles. However, eventhought there were multiple peptides on the quantum dot, the overallparticle polarity could not actually be reversed. These results help indetermining the level of charge a peptide would need to have, in orderto cause quantum dot to reverse charge.

Example 2—Detection of Pancreatic α-Chymotrypsin and Pancreatic Trypsinin 1×PBS Buffer Using a Net Charge Differentiating Peptide Substrate

In this experiment a 1 mg/ml (650 μM) stock solution of the NCDPSsubstrateAc-N-Asp-Gly-Asp-Ala-Gly-Tyr-Ala-Gly-Leu-Arg-Gly-Ala-Gly-diamino-ethyl-BodipyFL (SEQ ID NO:29) (MW 1537.4) was prepared by dissolving the peptide in1× phosphate buffered saline (PBS) at pH 7.8. Stock solutions fromlyophilized bovine pancreatic α-chymotrypsin (MW 25 kDa) and bovinepancreatic trypsin (MW 23.8 kDa) were prepared at 2 mg/ml (80 μMα-chymotrypsin and 84 μM trypsin) concentrations in 1 mM HCl. Bothproteases were serially diluted in 1 mM HCl in order to obtain thedesired concentrations. A solution of 486 μg/ml (316 μM) of theAc-N-Asp-Gly-Asp-Ala-Gly-Tyr-Ala-Gly-Leu-Arg-Gly-Ala-Gly-diamino-ethyl-BodipyFL peptide was prepared in 1×PBS (pH 7.8). Aliquots of 14.4 μl of theDCDPS peptide solution (1×PBS) were mixed with 1 μl aliquots of variousconcentrations of the proteases or 1 ul of 1 mM HCl for the negativecontrol, and then allowed to react for one hour. 6 μl samples containing3 μg (300 μM) of the substrate with protease concentrations ranging from500 nM (lanes 1-2), 100 nM (lanes 3-4), 50 nM (lanes 5-6), 30 nM (lanes7-8), 20 nM (lanes 9-10) or 0 nM (lanes 11-12) were loaded into thewells of a 4% high resolution agarose gel run in 0.5×TBE. The sampleswere electrophoresed at 80 V for 0.5 hr and then visualized directly bya BioDoc-It® System with a Model M-26 transilluminator (UVP, Upland,Calif.) at an excitation of 302 nm and through a SYBR® Green filter(passing 500-580 nm with peak transmission of 90% at 540 nm). The gelresults are shown in FIG. 20, and by inspection indicate a detectionlevel of about 20 nM for chymotrypsin and about 30 nM for trypsin in1×PBS. Gels were then quantified with a Storm 480 gel scanner usingImageQuant v5.2 (Molecular Dynamics, Sunnyvale, Calif.) (fluorescencemode, high sensitivity, 100 μm pixel size, 1000V photomultiplier tube)with a 450 nm excitation filter and a 520 nm long pass emission filter.The plotted results which are presented in FIG. 21 gave a detectionlimit of about 10 nM (0.02 activity units′/ml) for α-chymotrypsin and 20nM (8 activity units/ml) for trypsin in 1×PBS.

Example 3—Detection of Pancreatic α-Chymotrypsin and Pancreatic Trypsinin Human Plasma Using a Net Charge Differentiating Peptide Substrate

In this example a 1 mg/ml (650 μM) stock solution of the peptidesubstrate was prepared from lyophilizedAc-N-Asp-Gly-Asp-Ala-Gly-Tyr-Ala-Gly-Leu-Arg-Gly-Ala-Gly-diamino-ethyl-BodipyFL (MW 1537.4) (SEQ ID NO:29) by dissolving the peptide in 1×PBS at pH7.8. Stock solutions from lyophilized bovine pancreatic α-chymotrypsin(MW 25 kDa) and bovine pancreatic trypsin (MW 23.8 kDa) were prepared at2 mg/ml (80 μM α-chymotrypsin and 84 μM trypsin) concentrations in 1 mMHCl. Both proteases were serially diluted in 1 mM HCl in order to obtainthe desired concentrations. A solution of 486 μg/ml (316 μM) of theAc-N-Asp-Gly-Asp-Ala-Gly-Tyr-Ala-Gly-Leu-Arg-Gly-Ala-Gly-diamino-ethyl-BodipyFL peptide was prepared in un-diluted human plasma. Aliquots of 14.4 μlof the peptide plasma solution were mixed with 1 μl aliquots of variousconcentrations of the proteases or 1 ul of 1 mM HCl for the negativecontrol and then allowed to react for one hour. Samples of about 6 μleach containing 3 μg (300 μM) of the substrate with proteaseconcentrations that ranged from 500 nM (lanes 1-2), 100 nM (lanes 3-4),50 nM (lanes 5-6), 30 nM (lanes 7-8), 20 nM (lanes 9-10) or 0 nM (lanes11-12) were loaded into the wells of a 4% high resolution agarose gel in0.5×TBE. The samples were electrophoresed at 80 V for 0.5 hr and thenvisualized directly by a BioDoc-It® System with a Model M-26transilluminator (UVP, Upland, Calif.) at an excitation of 302 nm andthrough a SYBR® Green filter (passing 500-580 nm with peak transmissionof 90% at 540 nm). The gel results are shown in FIG. 22, and indicatedby inspection a detection level of about 20 nM for chymotrypsin andabout 20 nM for trypsin in plasma. Gels were then quantified with aStorm 480 gel scanner using ImageQuant v5.2 (Molecular Dynamics,Sunnyvale, Calif.) (fluorescence mode, high sensitivity, 100 μm pixelsize, 1000V photomultiplier tube) with a 450 nm excitation filter and a520 nm long pass emission filter. The plotted results which arepresented in FIG. 23 give a detection limit of about 10 nM (0.02activity units¹/ml) for α-chymotrypsin and 20 nM (8 activity units²/ml)for trypsin in 1× plasma.

Example 4—Detection of NCDPS Chymotrypsin and Trypsin Cleavage Fragmentsin Whole Blood

A 1 mg/ml stock solution ofAc-N-Asp-Gly-Asp-Ala-Gly-Tyr-Ala-Gly-Leu-Arg-Gly-Ala-Gly-diamino-ethyl-BodipyFL (SEQ ID NO:29) peptide substrate and various concentrations (2 mg/mlstock solution and serial dilutions of stock) of bovine pancreaticα-chymotrypsin and trypsin were prepared in the manner described in theabove examples. For detection of the cleavage products from reactionwith α-chymotrypsin, a solution of 486 μg/ml (316 μM) of theAc-N-Asp-Gly-Asp-Ala-Gly-Tyr-Ala-Gly-Leu-Arg-Gly-Ala-Gly-diamino-ethyl-Bodippeptide was prepared in 1×PBS, pH 7.8. In individual reaction tubes,14.4 μl aliquots of this peptide solution was mixed with 0.5 μl aliquotsof various concentrations of α-chymotrypsin (500 nM, 100 nM, 50 nM, 30nM, 20 nM or 0 nM) and then allowed to react for 1 hour. Heparinizedwhole rat blood was treated with an equal volume of 10× proteaseinhibitor cocktail and allowed to incubate for 1 minute. Each 14.9 μlreaction mixture of enzyme and substrate was then mixed with 7 μl ofprotease inhibitor-treated blood, forming a mixture containing 16% (v/v)blood Immediately after mixing the treated blood with the reactionmixture, 6 μl of each sample was loaded into the wells of a 4%high-resolution agarose gel (0.5×TBE) and electrophoresed at 80 V for0.5 hour. The gels were visualized and quantified as was described inthe above examples. For detection of the cleavage products by trypsin, asolution of 486 μg/ml (316 μM) of theAc-N-Asp-Gly-Asp-Ala-Gly-Tyr-Ala-Gly-Leu-Arg-Gly-Ala-Gly-diamino-ethyl-BodipyFL peptide was prepared in 1×PBS at pH 7.8. In individual reactiontubes, 7.2 μl aliquots of this peptide solution was mixed with 0.5 μlaliquots of various concentrations of trypsin (500 nM, 100 nM, 50 nM, 30nM, 20 nM or 0 nM) and then allowed to react for 1 hour. Heparinizedwhole rat blood was treated with 10× protease inhibitor cocktail asbefore and each 7.7 μl reaction mixture was mixed with 7 μl of proteaseinhibitor-treated blood, forming a final solution containing 24% (v/v)blood Immediately after mixing the treated blood with the reactionmixture, samples were electrophoresed, visualized, and then quantifiedas described above. The gel results for both chymotrypsin and trypsinare shown in FIG. 24, and give by inspection a an estimated detectionlimit of 20 nM (0.03 activity units¹/ml) for α-chymotrypsin and 50 nM(10 activity units²/ml) for trypsin. FIG. 25 shows the plotted resultsand also gives give by inspection a detection limit of 20 nM (0.03activity units¹/ml) for α-chymotrypsin and 50 nM (10 activity units²/ml)for trypsin in blood.

Example 5—Detection of Chymotrypsin/Trypsin Activity in Whole BloodUsing NCDPS Substrate

In this experiment 7 μl of 1× phosphate buffered saline (PBS) at pH 7.8or 7 μl of whole rat blood were mixed with the following: 7 μl of 1mg/mlAcetyl-N-Asp-Gly-Asp-Ala-Gly-Tyr-Ala-Gly-Leu-Arg-Gly-Ala-Gly-Bodipy FL(SEQ ID NO:26) in 1×PBS pH 7.8; 0.4 μl of 2 M CaCl₂; and 0.5 μl of thefollowing concentrations 2 mg/ml, 0.4 mg/ml, 0.08 mg/ml and 0 mg/ml ofhuman pancreatic α-chymotrypsin. The mixtures were incubated for 1 hourand then 6 μl of sample was loaded into the wells of a 4% highresolution agarose gel. The five wells on the left-hand side were loadedwith 1×PBS samples and the five wells on the right right-hand side wereloaded with the whole rat blood samples. Electrophoresis was performedfor 30 minutes at 80 V and then the gel was imaged using a BioDoc-It®System with a Model M-26 transilluminator (UVP, Upland, Calif.). Resultsfor the experiment are shown in FIG. 26A and FIG. 26B. The upper FIG.26A show the gel before the electric field was applied and the quenchingof fluorescent signal from NCDPS substrate by the blood samples canclearly be seen in the five wells on the left side. Thissensitivity-reducing quenching issue would occur for many of the currenttypes of fluorescent enzyme assays utilizing classical fluorogenicsubstrates. As a result, these latter methods typically requireconsiderable sample preparation, which is time-consuming and makes themeasurements less accurate, in order to recover fluorescent signal. Thelower FIG. 26B shows the appearance of cleavage products in the firstthree 1×PBS samples and in the blood samples. The results demonstratethe rapid separation of fluorescent signal from blood and subsequentrecovery (un-quenching) of fluorescent signal eliminating the need forsample preparation.

Example 6—Determination of Non-Specific Binding of Pancreaticα-Chymotrypsin/Trypsin Net Charge Differentiating Substrate

A 1 mg/ml solution of the substrateAc-N-Asp-Gly-Asp-Ala-Gly-Tyr-Ala-Gly-Leu-Arg-Gly-Ala-Gly-diamino-ethyl-BodipyFL (MW 1537.4) (SEQ ID NO:29) was prepared in 1×PBS, pH 7.8 and 7 μlaliquots of this solution were then each mixed with 1 μl of either 2mg/ml bovine pancreatic α-chymotrypsin or 1 mM HCl, as a negativecontrol. This reaction was allowed to proceed for 1 hour. Rat blood wasobtained as before and half of it was treated with an equal volume of10× protease inhibitor cocktail, while the other half was mixed with anequal volume of 1×PBS, pH 7.8. To each 8 μl solution of substrate (withor without α-chymotrypsin added), 14 μl of either untreated rat blood,protease inhibitor-treated rat blood, or 1×PBS, pH 7.8 was added andgently mixed. For samples with blood added, the final concentration ofblood was 32% (v/v). Samples were then electrophoresed and visualized inthe same way as described above. In order to compare data from bothgels, the samples containing only peptide in 1×PBS were split betweenthe two gels as a control. The results in FIG. 27 show that theuncleaved substrate in 1×PBS (unreacted with enzyme) that had migratedtoward the anode as a single band (see FIG. 27, No Enyzme—1×PBS—A band),now, after cleavage by α-chymotrypsin, migrates as two bands toward thecathode, representing a primary (see FIG. 27, Enzyme—1×PBS—HMC band) anda secondary (see FIG. 27, Enzyme—1×PBS—LMC band) α-chymotrypsin cleavageproduct. Comparing the reactions in 1×PBS (see FIG. 27, Enzyme/NoEnzyme—1×PBS—LMC/HMC bands) with the reactions in protease inhibitortreated-blood (see FIG. 27, Enzyme/No Enzyme—BLOOD+I—LMC/HMC bands),with and without α-chymotrypsin added, the data shows that there was nosignificant shift up or down of the SNR from the cathodic bands. Ifthere was significant non-specific binding altering the net charge ofthe uncleaved substrate or of the fluorescently labeled cleavageproducts, then there should be a significant shift of the SNR of thosebands. Therefore, this data shows that non-specific binding is notoccurring to any significant degree and hence does not affect theaccuracy of this detection scheme.

Example 7—Stacking Gel-Based Concentration of a Net ChargeDifferentiating Protease Substrates to Improve Sensitivity

A discontinuous gel was used in order to demonstrate a focusing of theα-chymotrypsin/tryspin substrateAc-N-Asp-Gly-Asp-Ala-Gly-Tyr-Ala-Gly-Leu-Arg-Gly-Ala-Gly-diamino-ethyl-BodipyFL (SEQ ID NO:29). A 7 mm wide 1% agarose gel surrounded by 2 mm wide20% T, 5% C polyacrylamide gels was created, with 0.5×TBE used for boththe gel casting buffer and running buffer. Samples of 0.5 mg/mlsubstrate in 0.5×TBE were loaded, in 6 μl aliquots, into wells formed inthe polyacrylamide gel and then electrophoreses at 300 V for 10 minutesin order to get the substrate to reach the barrier between the two typesof gel and focus. The results, shown in FIG. 28 demonstrate that after10 minutes the substrate migrated from the sample loading well, througha lower-density into higher-porosity agarose gel and then concentratedupon reaching a lower-porosity barrier formed by a higher-densitypolyacrylamide gel.

Example 8—High Voltage Electrophoresis in a High-Density PolyacrylamideCapillary Gel to Focus NCDPS to Improve Sensitivity

To a 1 mm O.D. capillary filled with 2 cm of a 26% T, 8% Cpolyacrylamide gel in 0.5×TBE, a 2 μl sample of 0.3 mg/ml of theAc-N-Asp-Gly-Asp-Ala-Gly-Tyr-Ala-Gly-Leu-Arg-Gly-Ala-Gly-diamino-ethyl-BodipyFL (SEQ ID NO:29) substrate was applied. In a running buffer of 0.5×TBEelectrophoresis was performed at 200 V (10 V/mm) for 5 minutes. Imagingwas performed with an epifluorescent microscope with a 2.5×, 0.07 NAObjective, a Bodipy TR Filter Set, and a Peltier thermal controlledHamamatsu Orca-ER CCD camera. The results, shown in FIG. 29, demonstratehigh concentration and focusing of the fluorescent substrate into 50 μmand 80 μm-wide bands after 5 minutes of electrophoresis. The 2 μl samplevolume injected into the 1 mm diameter capillary spans approximately a2.5 mm length of capillary, before the electric field was applied.Therefore, this concentration of the fluorescent signal into the twobands, totaling 130 μm of capillary length, represents more than a 20fold concentration of the signal.

Example 9—Detection of α-1 Insulin Receptor Cleavage Fragments in BloodUsing a Charge Changing Antibody Assay

In this example the net charge changing antibody method is used for thedetection of α-1 insulin receptor peptide cleavage fragments (fromMMP-9) directly in a whole blood sample. Into a 50 ul blood sample isadded a 10 ul aliquot (1×TBE buffer, pH 7.8) containing fluorescent (Ex490 nm and Em 510 nm) antibody 1 (for α-1 insulin receptor peptidecleavage fragment epitope A) at 100 ng/ml, and the non-fluorescentcationic antibody 2 (for α-1 insulin receptor peptide cleavage fragmentepitope B) at 100 ng/ml. The patient or test samples along with anegative control (normal blood) are allowed to react for about 15minutes, and then 10 ul of each sample is placed into the sample chamberof the focusing gel device. A DC electric field at 200 volts is nowapplied to the system for about five minutes. After the separation hasbeen achieved the anode (negative electrode) side of the focusing gel isscanned with an epifluorescent detector and the fluorescent signals (at510 nm) for the samples and control are detected and analyzed. Thepresence of any clinically relevant amounts of the α-1 insulin receptorpeptide cleavage fragments in the patient/test samples produces a higherfluorescent signal, and indicates the presence of MMP-9 which isdiagnostic for diabetes and associated inflammation.

VIII. References

-   1. Schmid-Schönbein, G. W. and T. E. Hugh, A new hypothesis for    microvascular inflammation in shock and multiorgan failure:    self-digestion by pancreatic enzymes. Microcirculation, 2005.    12(1): p. 71-82.-   2. Waxman, K., Shock: ischemia, reperfusion, and inflammation. New    Horiz, 1996. 4(2): p. 153-60.-   3. Wellen, K. E. and G. S. Hotamisligil, Inflammation, stress, and    diabetes. Journal of Clinical Investigation, 2005. 115(5): p.    1111-1119.-   4. Grimble, R. F., Inflammatory status and insulin resistance. Curr    Opin Clin Nutr Metab Care, 2002. 5(5): p. 551-9.-   5. Anselmi, A., et al., Myocardial ischemia, stunning, inflammation,    and apoptosis during cardiac surgery: a review of evidence. European    Journal of Cardio-Thoracic Surgery, 2004. 25(3): p. 304-311.-   6. Engler, R. L., G. W. Schmid-Schonbein, and R. S. Pavelec,    Leukocyte capillary plugging in myocardial ischemia and reperfusion    in the dog. American Journal of Pathology, 1983. 111(1): p. 98-111.-   7. Entman, M. L., et al., Inflammation in the course of early    myocardial ischemia. The FASEB journal, 1991. 5(11): p. 2529-2537.-   8. Ross, R., Atherosclerosis—an inflammatory disease. New England    Journal of Medicine, 1999. 340(2): p. 115.-   9. Schmid-Schonbein, G. W., S. Takase, and J. J. Bergan, New    advances in the understanding of the pathophysiology of chronic    venous insufficiency. Angiology, 2001. 52(1): p. S27-34.-   10. Suematsu, M., et al., The inflammatory aspect of the    microcirculation in hypertension: oxidative stress,    leukocytes/endothelial interaction, apoptosis.    Microcirculation, 2002. 9(4): p. 259-276.-   11. del Zoppo, G. J., Microvascular responses to cerebral    ischemia/inflammation. Annals of the New York Academy of    Sciences, 1997. 823(1): p. 132.-   12. del Zoppo, G. J., et al., Polymorphonuclear leukocytes occlude    capillaries following middle cerebral artery occlusion and    reperfusion in baboons. Stroke, 1991. 22(10): p. 1276-1283.-   13. Iadecola, C. and M. Alexander, Cerebral ischemia and    inflammation. Curr Opin Neurol, 2001. 14(1): p. 89-94.-   14. Jean, W. C., et al., Reperfusion injury after focal cerebral    ischemia: the role of inflammation and the therapeutic horizon.    Neurosurgery, 1998. 43(6): p. 1382-1396.-   15. Kontos, C. D., et al., Cytochemical detection of superoxide in    cerebral inflammation and ischemia in vivo. American Journal of    Physiology-Heart and Circulatory Physiology, 1992. 263(4): p.    1234-1242.-   16. Koistinaho, M. and J. Koistinaho, Interactions between    Alzheimer's disease and cerebral ischemia-focus on inflammation.    Brain Research Reviews, 2005. 48(2): p. 240-250.-   17. Schmid-Schönbein, G. W., Analysis of inflammation. Annu. Rev.    Biomed. Eng., 2006. 8: p. 93-151.-   18. Acosta, J. A., et al., Intraluminal pancreatic serine protease    activity, mucosal permeability, and shock: a review. Shock, 2006.    26(1): p. 3-9.-   19. Schmid-Schönbein, G. W., Mechanisms for cell activation and its    consequences for biorheology and microcirculation: Multi-organ    failure in shock. Biorheology, 2001. 38(2): p. 185-201.-   20. Schmid-Schönbein, G. W., et al., Pancreatic enzymes and    microvascular cell activation in multiorgan failure.    Microcirculation, 2001. 8(1): p. 5-14.-   21. Tierney, L. M., S. J. McPhee, and M. A. Papadakis, Current    medical diagnosis & treatment. 2002: Lange Medical    Books/McGraw-Hill.-   22. DeLano, F. A. and G. W. Schmid-Schonbein, Proteinase activity    and receptor cleavage. Mechanism for insulin resistance in the    spontaneously hypertensive rat: Hypertension 52 (2008), pp. 415-452-   23. Lee, S. W., et al., Alterations in peripheral blood levels of    TIMP-1, MMP-2, and MMP-9 in patients with type-2 diabetes. Diabetes    Research and Clinical Practice, 2005. 69(2): p. 175-179.-   24. Derosa, G., et al., Evaluation of metalloproteinase 2 and 9    levels and their inhibitors in diabetic and healthy subjects.    Diabetes and Metabolism, 2007. 33(2): p. 129-134.-   25. E. G. DelMar, C. Largman, J. W. Brodrick, M. C. Geokas, A    sensitive new substrate for chymotrypsin, Anal. Biochem. 99 (1979)    316-320.-   26. M. Zimmerman, B. Ashe, E. C. Yurewicz, G. Patel, Sensitive    assays for trypsin, elastase, and chymotrypsin using new fluorogenic    substrates, Anal. Biochem. 78 (1977) 47-51.-   27. E. Anderson, K. W. C. Sze, S. K. Sathe, New colorimetric method    for the detection and quantitation of proteolytic enzyme    activity, J. Agric. Food Chem. 43 (1995) 1530-1534.-   28. L. J. Jones, R. H. Upson, R. P. Haugland, N.    Panchuk-Voloshina, M. Zhou, Quenched BODIPY dye-labeled casein    substrates for the assay of protease activity by direct fluorescence    measurement, Anal. Biochem. 251 (1997) 144-152.-   29. D. Stockholm, M. Bartoli, G. Sillon, N. Bourg, J. Davoust, I.    Richard, Imaging calpain protease activity by multiphoton FRET in    living mice, J. Mol. Biol. 346 (2005) 215-222.-   30. Y. Zhang, C. Haskins, M. Lopez-Cruzan, J. Zhang, V. E.    Centonze, B. Herman, Detection of mitochondrial caspase activity in    real time in situ in live cells, Microsc. Microanal. 10 (2004)    442-448.-   31. L. M. Levine, M. L. Michener, M. V. Toth, B. C. Holwerda,    Measurement of specific protease activity utilizing fluorescence    polarization, Anal. Biochem. 247 (1997) 83-88.-   32. S. Z. Schade, M. E. Jolley, B. J. Sarauer, L. G. Simonson,    BODIPY-alpha-casein, a pH-independent protein substrate for protease    assays using fluorescence polarization, Anal. Biochem. 243 (1996)    1-7.-   33. D. E. Kleiner, W. G. Stetlerstevenson, Quantitative zymography:    detection of picogram quantities of gelatinases, Anal. Biochem.    218 (1994) 325-329.-   34. White, D., J. Stevens and J. Shultz, PepTag protease assay: a    simple and rapid method for detection of very low amounts of    protease, Promega Notes Magazine, 344, November 1993, p2.-   35. U.S. Pat. No. 5,580,747, Dec. 3, 1996, J. W. Shultz and D. H.    White-   36. U.S. Pat. No. 6,942,778, Sep. 13, 2005, S. Jalali et al.-   37. WO 2005/0666359 A1, Jul. 21, 2005, H. Sun et al

What is claimed is:
 1. A method of detecting a biomolecule in a bodilyfluid sample, said method comprising the steps of: (i) contacting abodily fluid sample with a first detection antibody comprising adetectable moiety and a second positively charged antibody to form adetectable positively charged biomolecule conjugate; (ii)electrophoretically separating said detectable positively chargedbiomolecule conjugate from negatively charged endogenous materialpresent in said bodily fluid sample; and (iii) detecting said detectablepositively charged biomolecule conjugate thereby detecting thebiomolecule in the bodily fluid sample.
 2. The method of claim 1,wherein said bodily fluid sample is a blood sample or a blood plasmasample.
 3. The method of claim 1, wherein said biomolecule is an enzyme,a proenzyme, a protein, an antibody, a peptide, a peptide cleavagefragment, a hormone, a virus, a small biomolecule or a drug molecule. 4.The method of claim 1, wherein said detectable moiety is a fluorophore,a quantum dot, a fluorescent nanoparticle, a dendrimeric nanoparticle, ametallic nanoparticle, a chemiluminescent label, a electrochemical labelor an oxidation/reduction label.
 5. The method of claim 4, wherein saiddetectable moiety is a fluorophore.
 6. The method of claim 4, whereinsaid detectable moiety is a chemiluminescent label.
 7. The method ofclaim 1, wherein said first detection antibody comprises an antibodythat is conjugated at the Fc region to a fluorophore or fluorescentprotein, polymer or a dendrimer.
 8. The method of claim 7, wherein saidsecond positively charged antibody comprises an antibody conjugated atthe Fc region to a non-fluorescent polymer or dendrimer.